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We are the tech transfer platform for the life science tools.

We do product development side by side with the researchers who design and use innovative R&D tools

Discover our portfolio of innovative R&D tools 

Products

SpheroTribe - The all-in-one kit for easy 3D cell culture

SpheroTribe provides a complete toolkit for generating reproducible 3D cell structures (spheroids, organoids) without restrictions of cell type.

The kit includes:
– 5X methylcellulose solution
– U-bottom 96-well plates
– pipette tips with a large opening of 200µL

Size/content Catalog Number
10x 96-well plates (flat bottom) 2x tip boxes 1 methylcellulose bottle TDA-SPK-KIT
1x 96-well plates (flat bottom) 20 tips 2,5 mL of methylcellulose TDA-SPK-MINIKIT
1 methylcellulose bottle TDA-SPK-2-25

SpheroTribe provides a simple toolkit to generate consistent and robust 3D cell structures. Simply dilute the SpheroTribe solution into your culture medium of choice, watch your cells turn into uniformly sized 3D spheroids and collect them for your downstream assays.

Once diluted in your culture medium of choice, our concentrated polymer-based solution increases the medium viscosity favouring cell-cell contacts. SpheroTribe offers a simple method to generate homogeneous 3D cell structures with increased control over their size and shape, which can be easily handled and washed for downstream experiments.

SpheroTribe is particularly useful to boost aggregation when working with challenging cells, minimize variability between samples and improve the consistency of your migration/invasion assays, immunostaining, drug screening or in vivo implantation experiments.

Kit description

In addition to the SpheroTribe solution, the full kit also includes U-bottom plates and wide-opening tips so you have everything you need to get started with your 3D cell culture experiments.

25mL kit contents:
– 25mL of 5X methylcellulose solution
– 10x U-bottom 96-well plates
– 2x racks of 96 pipette tips (200µL) with a large opening
2,5mL kit contents:
– 2.5mL of 5X methylcellulose solution
– 1x U-bottom 96-well plate
– 20 pipette tips (200µL) with a large opening

Storage

Recommendation for the methylcellulose solution:
4°C for (at least) 6 months.

Cell aggregation booster

SpheroTribe provides a gel-like scaffold that favors cell-cell contacts by increasing medium viscosity. It was shown to generate compact spheroids mimicking solid tumors, improve spheroid formation with some of the most challenging cells and to speed up stem cell-derived organoid formation.

Increased homogeneity

By maximizing cell aggregation, SpheroTribe promotes the formation of unique & uniformly sized spheroids allowing for consistent assays (growth, invasion, immune infiltration, in vivo injection, etc).

Easy to use

No need to work on ice, have access to sophisticated equipment of expertise. With SpheroTribe, you have everything on hand to easily grow & handle your spheroids.

Universal

The SpheroTribe solution is composed of methylcellulose, a biologically inert compound dissolved in basal culture medium without any proteins, lipids or growth factors. You can dilute it in any culture medium of your choice, and add additional compounds as desired (i.e. serum, antibiotics, differentiation factors, etc).

Applications:

So far, SpheroTribe has been successfully used for spheroid/organoid formation with the following cell types:

Patient-derived stem-like glioblastoma cells (GB P3 and BL13), human glioblastoma cell lines (U87 & T98G), HeLa, human vaginal mucosal melanoma (HMV-II), human primary colorectal cancer cells, human breast cancer cells (MDA-MB 231), human induced pluripotent stem cells, monkey kidney fibroblast-like cell line (COS-7), primary neurons from rat embryos (E18) & murine melanoma cells (B16F10).

Experimental assays:

Once spheroids have grown to your desired size, you can use them for any kind of assay according to your regular workflow. The SpheroTribe solution can be readily washed off, leaving a spheroid available for other tests at any stage of your protocol.

Example of in situ assays you can perform directly on the U-bottom plate supplied:

  • Live imaging
  • Growth/proliferation studies
  • Toxicity studies

Examples of downstream assays that might require transferring spheroids to other vessels:

  • Invasion & migration assays
  • Immunostaining
  • Biochemical assays
  • Immune infiltration assays
  • In vivo implantation

For more guidance on downstream assays, check out our FAQ section containing useful tips & example protocols.

Uniform patient-derived glioblastoma spheroids generated using the SpheroTribe kit and visible by the naked eye after 4 days of culture
SpheroTribe improves the formation of unique & circular human glioblastoma spheroids

Human glioblastoma U87 cells were cultured in DMEM with or without SpheroTribe
in U-bottom plates and imaged after 2 days (4X magnification) and 6 days (10X magnification)

Patient-derived glioblastoma spheroid formed with SpheroTribe invading a 3D collagen-I matrix

Glioblastoma spheroids were included into a collagen matrix after being cultured in medium added with SpheroTribe solution for 4 days.
Images were taken immediately after (left) or 24 hours after (right) inclusion in the collagen matrix.
Image credits: (c) Thomas Daubon, 2023.

Immune infiltration of B16F10 spheroids after immune checkpoint blockade

A. 10,000 B16F10 cells were grown for 6 days as spheroids using SpheroTribe. B. 100,000 PBMC from murine spleen were activated with IL-15 (40 ng/mL) [1], incubated with anti-PD1 (10 µg/mL) for 1h and added on B16F10 spheroids for 3 days.
Graph shows flow cytometry quantification of differential lymphocyte infiltration after spheroid dissociation according to treatment. N=4. Mann-Whitney U Test, p-value<0.05. [1] https://doi.org/10.3389/fonc.2022.898732.
Image credits: Guillaume Mestrallet, PhD – Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, US – 2023. @GMestralletPhD

SpheroTribe improves cardiac organoid formation by boosting hiPSC aggregation

Human iPSCs were seeded in ULA plates in presence (left) or absence (right) of SpheroTribe solution (T=0) and cultured for 3 days following a self-assembling human heart organoid differentiation protocol [1].

Image credits: Aitor Aguirre, Ph. D. – Michigan State University, US

[1] Lewis-Israeli, Y.R., Wasserman, A.H., Gabalski, M.A. et al. Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease. Nat Commun 12, 5142 (2021). https://doi.org/10.1038/s41467-021-25329-5

SpheroTribe improves spheroid formation in agarose micro-wells

U-87 glioblastoma cells were seeded at 1,000 cells per well in round-bottom wells molded in agarose using a Stampwell U-shape (Idylle) in full culture medium without (right) or added with SpheroTribe (left). After 3 days, pictures were taken and number of cell aggregates per well were quantified from 64 independent wells and associated standard deviation (SD) values were calculated.

SpheroTribe promotes and maintains cell aggregation in COS-7 monkey kidney fibroblast-like cells

COS-7 cells were cultured in non-tissue culture treated U-bottom plates for 4 days in presence (up)
or absence (bottom) of the SpheroTribe concentrate. Scale bar = 500 µm

SpheroTribe promotes and maintains cell aggregation in HeLa cells

HeLa cells were cultured in non-tissue culture treated U-bottom plates for 4 days in presence (up)
or absence (bottom) of the SpheroTribe concentrate. Scale bar = 500 µm

How to use SpheroTribe:

Video protocol

Read the full protocol

How to use SpheroTribe in pictures

Need advice for your downstream assays? Check out our FAQ section for published protocols of immunostaining, invasion, migration, proliferation assays & in vivo spheroid implantation.

General enquiries

How does SpheroTribe work?

SpheroTribe relies on the use of methylcellulose, a crowding agent that is well-known to drive efficient cell aggregation into spheroids. Once diluted in culture medium, methylcellulose increases the viscosity of the medium, creating a gel-like environment that restrict cell movement and encourages cell-cell contacts for the formation of compact spheroids.

Can SpheroTribe affect my cell physiology or subsequent assays?

The SpheroTribe solution is composed of methylcellulose, a derivative of cellulose that is known to be biologically-inert, i.e. to neither actively contribute to biological responses or interfere with it. It is notably used in various biofabrication tools, including bioinks. In addition, SpheroTribe allows you to culture cells without exogenous ECM components, avoiding the known bias they may have on cell signaling. If needed, SpheroTribe can be readily washed off at any stage of the protocol to leave spheroids available for subsequent assays.

Which cells has SpheroTribe been used with?

So far, SpheroTribe has been successfully used to generate spheroids/organoids with the following cell types:

Patient-derived stem-like glioblastoma cells (GB P3 and BL13), human glioblastoma cell lines (U87 & T98G), HeLa, human vaginal mucosal melanoma (HMV-II), human primary colorectal cancer cells, human breast cancer cells (MDA-MB 231), monkey kidney fibroblast-like cell line (COS-7), primary neurons from rat embryos (E18) & murine melanoma cells (B16F10), human induced pluripotent stem cells (hiPSCs)-derived organoids.

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Is there a particular medium I need to use SpheroTribe with?

The SpheroTribe concentrated solution is made up in basal medium and does not contain any proteins, lipids or growth factors. You can dilute it in any culture medium of your choice, and add other compounds as desired (i.e. serum, antibiotics, etc).

How many experiments can I perform with one SpheroTribe kit?

One SpheroTribe kit contains everything you need to grow up to 960 unique spheroids. Total number of spheroids generated with one kit may vary depending on the concentration of SpheroTribe solution used for cell aggregation, total spheroid growth duration and frequency of medium renewal.

Can I buy the methylcellulose solution alone?

Yes, it is also possible to buy the methylcellulose solution alone and use it to complement your current method. Please contact us and we will send a dedicated quote.

Protocol of use

How long will it take to grow spheroids with SpheroTribe?

The total time to initiate 3D cell culture with SpheroTribe should take no more than 30 minutes. After that, optimal culture duration to achieve desired spheroid size will vary depending on your cell proliferation rate and targeted application. As a general rule, compact & homogeneous spheroids are usually formed after 3 days of culture.

How many cells should I seed in each well?

Optimal seeding density can vary greatly depending on the cell type, proliferation rate and targeted application. You will most probably need to start with a couple of optimization tests to find out which density will best suit your needs. As a general rule, we recommend using 10,000 to 20,000 cells per well for primary cells and 2,000 to 7,000 cells per well for tumoral/immortalized cell lines.

How big will my spheroids get?

Spheroid sizes can vary greatly depending on cell type characteristics, initial density and culture conditions used. To help you select optimal conditions, here are a couple of examples of sizes reached for different cell types and seeding densities:

Do you have specific recommandations for downstream assays?

A variety of downstream assays have been performed with spheroids generated with SpheroTribe. We only provide a detailed protocol for the formation of spheroids, as this is what SpheroTribe is designed for. However, if needed, you can check out the following publications describing protocols for various assays using glioblastoma spheroids generated with SpheroTribe. These can easily be applied to other cell types:

How can I make sure my spheroids will be unique and uniform ?

The SpheroTribe kit has been shown to generate unique & uniform spheroids with most cell types tested. Yet, when working with particularly challenging cells, here are a few tips to keep in mind to maximize your chance to obtain unique & uniform spheroids in each well:

  • Starting from a single-cell suspension: the presence of cell clumps in your initial cell suspension at the time of seeding can compromise the obtention of unique & uniform spheroids. When starting from isolated primary cells, we recommend that you carefully dissociate cells using filters or columns if needed.
  • Pre-heating the SpheroTribe solution at 37°C  before use: lower temperatures will make it more viscous, potentially favorising the formation of multiple cell aggregates.
  • If the above advice are not sufficient, centrifuging the plate at 300g for 3 minutes can help with spheroid formation.

Any other questions? Please contact us

Chitozen - Functionalized coverslips for bacteria live imaging

Chitosan-coated coverslips for live bacteria imaging

​Chitozen is a chitosan-coated coverslip immobilizing bacteria for microscopy without inducing bacteriostatic effects. It is compatible with 6-channel sticky slides for flow experiments. It is very helpful if you want to:

  • Image bacteria both still and alive under the microscope
  • Maintain bacteria in a same focal plane for imaging while preserving their physiology
  • Renew culture medium, change growth condition (e.g. antibiotics, chemicals, inhibitors) during the experiment and directly observe, in real-time, the bacteria new comportment under the microscope

Applications

Assay compatibility:
> Fluorescence
> Co-cultures (bacterial predators, immune cells)
> Addition of external factors (e.g. antibiotics, chemicals, inhibitors)
> Static or dynamic conditions
Imaging modes:
> phase-contrast,
> epifluorescence,
> confocal,
> super-resolution microscopy,
> atomic force microscopy (AFM) – documentation & example pictures available on request
Experimental outputs:
> Behavioural changes: growth, elongation, cell division, fitness, colony/biofilm formation, etc
> Single molecule imaging
Chitozen is efficient with the following bacteria:
> E. coli
> Bacillus subtilis
> Caulobacter crescentus
> Corynebacterium glutamicum
> Helicobacter pylori
> Mycobacterium smegmatis
> Myxococcus xanthus
> Pseudomonas aeruginosa
> Pseudomonas fluorescens
> Salmonella
> Staphylococcus aureus
> Vibrio cholerae
This list is updated regularly according to feedback provided by researchers who use Chitozen.

Kit contents

Chitozen coverslips are compatible with 6-channel sticky slides to work in a closed system in static or dynamic flow conditions. Order you coverslips alone if you need to work in an open system (i.e. for AFM imaging).

5-coverslips only*
> 5x standard (25 x 75 mm) chitosan-coated coverslips

5-coverslips with microfluidic channels
> 5x standard (25 x 75 mm) chitosan-coated coverslips
> 5x bottomless 6-channel sticky slides

(Optional) centrifugation pack: the Chitozen coverslips can be centrifuged using a rack compatible with standard microscope slide (25mmx75mm) dimensions. If needed, add our centrifugation pack to your Chitozen kit. It includes:
> µ-Slide Microscopy Rack (ibidi
> Magnetic Lid for Microscopy Rack
> Clamp & adapter for sticky slides

*If using the Chitozen coverslips alone, separate wells can be created manually by using a sealing glue or ​Stencell​ silicon chambers. We can provide these 2 products for you. ​Contact us​ for more information.

For custom orders (i.e. alternative coverslip amounts), please contact us.

Lifetime: up to 12 months at room temperature, shielded from direct sunlight

E. coli monolayers on Chitosan

© 2019 Tréguier et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Growth and division of E. coli on Chitozen, in LB medium

Credit: Amandine Desorme, LCB – CNRS, 2021

Visualization of Pal mCherry at septum in E. coli (W3110 Pal mCherry), by 3D SIM microscopy, in M9 medium and using Chitozen.

Credit: Amandine Desorme, LCB – CNRS, 2021

Observation of vesicles at septum during cell division of mutant E. coli (W3110 tolR – Palmcherry),
in LB 1/2 medium, using Chitozen.

Peptidoglycan is labeled with the green fluorophore BADA.

Credit: Amandine Desorme, LCB – CNRS, 2021

Live cell imaging of E. coli in response to drug addition on Chitozen slides

AB1157 E. coli expressing DNA marker HU-mCherry were imaged at 37oC with perfusion of ½ LB at a flow rate of 2 ml/min. Drug X was added at time point 35 minutes and removed at time point 60 minutes.

Credit: Emily Helgesen – Oslo University Hospital – 2022

Live cell imaging of E. coli expressing a protein associated with DNA replication on Chitozen slides

AB1157 E. coli expressing SeqA-YFP (green), a protein associated with DNA replication, were imaged at 37°C over 60 minutes without perfusion of medium

Credit: Emily Helgesen – Oslo University Hospital – 2022

Effects of cell division inhibitor cephalexin on E. coli growth cultured on Chitozen slides

E. coli BW25113 cells were imaged at 37°C with perfusion of M9 medium at a flow rate of 0.05 mL/min.
Credit: Bianca Sclavi – 2021

E. Coli spheroblasts bound to Chitozen

E. coli MG1655 with chromosomally encoded HU-eGFP under the native promoter were imaged with perfusion at a flow rate of 0.05 mL/min.

Credit: Itzhak Fishov – Ben-Gurion University of the Negev, 2022

How to use Chitozen coverslips:

Read the full protocol

Video protocol

Find below the advice & tips for Chitozen use:

Everspark - A long-lasting buffer for dSTORM

Everspark buffer at 100 mM MEA in Tris buffer
10 vials containing 450 μL each (total volume 4,5 mL)

Description Catalog Number
Everspark 1.0 KMO-ETE-450
Everspark 2.0 KMO|ETE+|450-10

Discover the long-lasting Everspark blinking buffer series for prolonged blinking in dSTORM microscopy. No more rushing your imaging. Mount your sample, and get stable blinking for weeks. ​​
NEW ! Discover the newly-developed Everspark 2.0 also compatible with green fluorophores (i.e. AF488) for improved resolution in 3-colour dSTORM microscopy.

Everspark 1.0

Compatible fluorophoresyellow to far-red 
Validated fluorophores include JF 549DL 550CF 555, CF 568, AF 568,  JF 646, AF 647CF 647, Atto 647N, DL 650CF 680 & Cy5

Multicolor imaging: +

Blinking stability (after mounting): up to 2 months

Blinking events: +

Everspark 2.0 

Compatible fluorophoresgreen, yellow & far-red 
Validated fluorophores include AF 488MemBright 488FITC, FAM, Spy555CF 568 & AF 647

Multicolor imaging : +++

Blinking stability (after mounting): up to 3.5 months

Blinking events: +++

Kit description:

10 vials with 450 µL of Everspark buffer at 100 mM MEA in Tris.
1 vial per experiment.
Vials lifetime: Up to 6 months in a closed pouch (eg unmounted).

Laser requirements: for green fluorophore imaging (Everspark 2.0 only), we recommend using a 488 laser >200mW, ideally 500mW for optimal results.

Co-localisation studies using three-colour 3D dSTORM imaging with Everspark 2.0

Centrosomal proteins Cep152, rootletin and PCM labelled with AF647, CF568 & AF488 respectively were imaged on a Vutara VXL (Bruker) inEverspark 2.0 buffer before 3D dSTORM reconstruction
Credits: Karine Monier, INMG-PGNM, Lyon

Co-localisation studies using three-colour 3D dSTORM imaging with Everspark 2.0

Centrosomal proteins Cep152, rootletin and PCM labelled with AF647, CF568 & AF488 respectively were imaged on a Vutara VXL (Bruker) in Everspark 2.0 buffer before 3D dSTORM reconstruction
Credits: Karine Monier, INMG-PGNM, Lyon

Green-channel dSTORM imaging of AF488 fluorophore using Everspark 2.0 buffer

AF488-coated beads were mounted in Everspark 1.0 or Everspark 2.0 buffer and imaged on a Vutara VXL (Bruker) before 3D dSTORM reconstruction.
Credits: Karine Monier, INMG-PGNM, Lyon

Long-term imaging with Everspark 1.0

Left: Two centrosomes imaged the same Day (D0) and 7 days after mounting (D7) on the same slide stored in the dark at 4°C. The typical 450 nm donut-like structure is visualised using a colour-coded scale encoded with the IGOR software, where each point appears as a function of its localisation precision (5 to 60 nm; inverted rainbow colour scale). Labeling: Distal-appendages detected by immunofluorescence with AF647 in RPE-1 cells.
Right: The number of blinking events per centrosome and the median of the localization precision in nm are presented for each serie of 50,000 images recorded at D0 and D7 (left).
Credits: Camille Fourneaux & Karine Monier, CRCL, Lyon

Donut-like structure of in-cellulo mature centrosome reconstructed after dSTORM imaging in Everspark 1.0 buffer

Each point is represented by its centroid (purple points) and its gaussian width (white). Intensity profil is displayed on the right with the measurement from peak to peak, in agreement with the size of the distal appendage crone. Labeling: distal-appendages detected by immunofluorescence with AF647 in RPE-1 cells.

Credits: Corentin Rousset, Karine Monier, CRCL Lyon

Everspark technology has been intially developed by Karine Monier, Arnaud Favier and Christophe Place and published in Scientific Reports:
Provost, A., Rousset, C., Bourdon, L. et al. Innovative particle standards and long-lived imaging for 2D and 3D dSTORM. Sci Rep 9, 17967 (2019). https://doi.org/10.1038/s41598-019-53528-0

Publications:

Dia2 formin controls receptor activity by organizing plasma membrane lipid partitioning at the nanoscale
Changting LI, Yannick HAMON, David MAZAUD, Pamela GONZALEZ TRONCOSO, Marie DESSARD, Hai-tao HE, Christophe LAMAZE, and Cedric M. BLOUIN. bioRxiv 2023.12.15.571857; doi: https://doi.org/10.1101/2023.12.15.571857

Nanoscale engagement and clusterization of Programmed death ligand 1 (PD-L1) in the membrane lipid rafts of Non-Small Cell Lung Cancer cells
Martina Ruglioni, Simone Civita, Tiziano Salvadori, Sofia Cristiani, Vittoria Carnicelli, Serena Barachini,  Iacopo Petrini, Irene Nepita, Marco Castello, Alberto Diaspro, Paolo Bianchini, Barbara Storti, Ranieri Bizzarri,  Stefano Fogli and Romano Danesi   bioRxiv 2022.08.09.503318; doi: https://doi.org/10.1101/2022.08.09.503318

HIV-1 diverts actin debranching mechanisms for particle assembly and release in CD4 T lymphocytes
Rayane Dibsy, Erwan Bremaud, Johnson Mak, Cyril Favard, Delphine Muriaux
Nat Commun. 2023 Oct 31;14(1):6945. doi: 10.1038/s41467-023-41940-0

Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin
Fabre L, Rousset C, Monier K, Da Cruz-Boisson F, Bouvet P, Charreyre MT, Delair T, Fleury E, Favier A. Biomacromolecules. 2022 May 12. doi: 10.1021/acs.biomac.1c01706. PMID: 35549176

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.
Michalik S, Siegerist F, Palankar R, Franzke K, Schindler M, Reder A, Seifert U, Cammann C, Wesche J, Steil L, Hentschker C, Gesell-Salazar M, Reisinger E, Beer M, Endlich N, Greinacher A, Völker U. Haematologica. 2022 Apr 1;107(4):947-957. doi: 10.3324/haematol.2021.280154. PMID: 35045692

Metabolic biorthogonal labeling and dSTORM imaging of peptidoglycan synthesis in Streptococcus pneumoniae
Jennyfer Trouve, Oleksandr Glushonkov and Cecile Morlot
Star Protocols, December 13, 2021. doi: 10.1016/j.xpro.2021.101006

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.
Greinacher A, Selleng K, Palankar R, Wesche J, Handtke S, Wolff M, Aurich K, Lalk M, Methling K, Völker U, Hentschker C, Michalik S, Steil L, Reder A, Schönborn L, Beer M, Franzke K, Büttner A, Fehse B, Stavrou EX, Rangaswamy C, Mailer RK, Englert H, Frye M, Thiele T, Kochanek S, Krutzke L, Siegerist F, Endlich N, Warkentin TE, Renné T.
Blood. 2021 Dec 2;138(22):2256-2268. doi: 10.1182/blood.2021013231. PMID: 34587242

Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres.
Szikora S, Novák T, Gajdos T, Erdélyi M, Mihály J. Bio Protoc. 2020 Jun 20;10(12):e3654. doi: 10.21769/BioProtoc.3654. eCollection 2020 Jun 20. PMID: 33659324

SpheroRuler - Calibration beads for SMLM microscopy

Calibration beads for super-resolution microscopy 1 vial of 50 µL of a SpheroRuler suspension in PBS pH 7.4

Description Catalog Number
10x 96-well plates (flat bottom) 2x tip boxes 1 methylcellulose bottle TDA-SPK-KIT
1x 96-well plates (flat bottom) 20 tips 2,5 mL of methylcellulose TDA-SPK-MINIKIT
1 methylcellulose bottle TDA-SPK-2-25

SpheroRuler is a monodispersed suspension of 1µm diameter spheres coated with 647-fluorophores giving a stable blinking in SMLM microscopy. Their consistent size and geometry make them very practical and reliable standards to assess the accuracy of your x-y or z measurements, 3D reconstruction methods or your image quality. Monodispersed and immobile in solution, they can also be used as rulers, for drift correction or as guides to help localize features on your biological samples.

Kit description: 1 vial of 50 µL of a SpheroRuler suspension in PBS pH 7.4.
Allows 10 experiments when using the recommended 5µL volume in 35mm glass-bottom dishes.

Concentration: 7.10exp8 particles per mL.

Stability: up to 7 months when stored at 4°C.

Dye: 647-fluorophore (far-red fluorescence).

Compatible with: dSTORM, SRRF, Airyscan confocal, confocal, SEM

The SpheroRuler family is expanding !

Following the success of SpheroRuler beads, we are thrilled to introduce some brand-new SpheroRulers of various sizes and colours that are now ready to join our catalog:

Interested in one or several of them? Please contact us

One SpheroRuler bead imaged using different microscopy techniques
SpheroRuler beads reconstructed using SRRF-Stream microscopy

(A, B). Pictures of SpheroRuler beads acquired in wide-field (A) and SRRF-Stream super-resolution (B) imaging. Scale bar = 5µm
(C, D) Local enlargements of A and B pictures respectively.
Scale bar = 1µm
(E) Fluorescence intensity distributions along the solid lines in C and D.

Credits: Yao Baoli – Xi’an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, 20lksehg

SpheroRuler beads as a benchmarking tool to assess the impact of different imaging modes on resolution and image quality

SpheroRuler beads were imaged on a confocal microscope with a 63X oil objective.

Credits: Kseniya Korobchevskaya – Institute of Developmental & Regenerative Medicine, University of Oxford, UK

2D and 3D dSTORM images of a SpheroRuler bead acquired with a Leica GSD system

Credits: Lydia Y Li, Nabi Lab, The University of British Columbia, 2023

How to use Spheroruler fluorescent beads:

Protocol overview in 4 steps:

What are SpheroRuler beads made of?

The SpheroRuler beads are 1μm-diameter polymer particles surrounded by 647-fluorophores covalently anchored to their surface. Beads will be visible as hollow rings or spheres when reconstructed in 2D or 3D SMLM experiments respectively

Which types of imaging is SpheroRuler compatible with?

SpheroRuler beads are coated with 647-fluorophores giving a stable blinking in SMLM microscopy, and have been initially developed for dSTORM imaging. Since then, they have also been successfully used in SFFR, SEM, confocal, Airyscan confocal and SEM microscopy.

How accurate is the SpheroRuler bead size?

The spherical particles making up Spheroruler beads have been selected based on very good monodispersity properties. The accuracy and reproducibility of the bead diameter have been characterized by SEM on 25 independent microspheres and showed a standard deviation of 1 +/- 0.05μm.

What is included in the SpheroRuler kit? Is there anything I need that is not provided?

The SpheroRuler kit contains a 50μL suspension of SpheroRuler beads. All you need to have on your side is some blinking buffer, coverslips and imaging vessels of your choice.

How many experiments can I carry out with one SpheroRuler kit?

One kit contains a 50μL suspension of SpheroRuler beads, allowing for 10 experiments when using the recommended 5μL volume in 35mm glass-bottom dishes.

Can I use it alongside my biological samples?

Yes, SpheroRuler beads are resuspended in PBS and can be loaded together with biological specimens (cells, tissue sections, etc).

How long can I store SpheroRuler for?

Yes, SpheroRuler beads are resuspended in PBS and can be loaded together with biological specimens (cells, tissue sections, etc).

How long can I store SpheroRuler for?

The SpheroRuler suspension is stable for at least 7 months when stored at 4°C.

Which object should I retrieve after reconstruction?

In 2D, you should retrieve a hollow circle of 1μm diameter. In 3D, you should retrieve a hollow sphere of 1μm diameter.

Is there any specific reconstruction algorithm I should use?

SpheroRuler beads are highly fluorescent beads coated with a high density of fluorophores. We recommend using a “multi-emitter / high density” rather than a “single-emitter / low density”, algorithm, to guarantee an efficient localization of individual blinking events. Although the available options will vary on each imaging system used, an example of algorithm that was successfully used is the “Account for overlap” function on the ZEN software (Zeiss).

How should fluorophore thickness be taken into account in the diameter measurement?

Fluorophores are directly coated on the bead surface without linkers or antibodies, and their thickness is therefore negligible compared to the measured diameter. The apparent thickness of the fluorophore ring will depend on the resolution of your imaging system (i.e. around 160-200nm when measured in dSTORM). The external periphery of the beads should be taken into account when measuring diameter.

I am not retrieving a perfectly spherical shape when reconstructing the beads in 3D. Is it normal?

Although 2D reconstructions of SpheroRuler beads should be accurately circular, obtaining an elongated shape in z is a common artefact that will depend on the system used for imaging and for 3D reconstruction (biplane reconstruction, astigmatism, etc). The measured diameter in z and its distance from the actual 1μm can therefore be used as a robust indicator to evaluate the fidelity of the 3D reconstruction for a given system. As an example, measured z diameter was 1.3 μm when tested on a Vutara VXL system using biplane imaging.

How can I maximize the resolution and fidelity of my reconstruction?

The SpheroRuler beads are coated with a high density of fluorophores. A few tips that will facilitate the SpheroRuler reconstruction:

– Make sure you perform a “pumping” step by illuminating with ultra-high power (i.e. laser 80% for less than 30s) before starting the acquisition in order to improve the localization precision.

– Use a “multi-emitter / high density” algorithm, or any equivalent, to guarantee an efficient localization of individual blinking events.

– If still not sufficient to efficiently reconstruct the spheres, or if such algorithm is not available, using a PSF filter to eliminate out-of-focus events or close double emitters should refine the localisation precision and make the peripheral crown more visible, therefore improving the reconstruction fidelity.

Actiflash - A photoinducible protein activator

Photoinducible protein activator (powder)

A technology developed by ​Ludovic Jullien​, ​Isabelle Aujard ​ and ​Thomas Le Saux​ – ENS Paris, France

Size Catalog Number
5 mg LJU-CIN-5
50 mg LJU-CIN-50

​Actiflash is a small steroid ligand that activates engineered proteins upon light induction. It can be used to precisely control protein activity down to the single cell level in live cell cultures and animals.

How does it work?

Actiflash is a tamoxifen-like caged-inducer called cyclofen. Upon UV-light illumination, optical-induced uncaging of Actiflash quickly releases proteins fused to the modified estrogen receptor ligand-binding domains ERT2. This allows them to translocate into the nucleus, activate transcription (using Gal4-UAS system) or induce recombination (using the Cre-lox one).

The protein expression can thus be simply controlled in time and space thanks to UV-light!

Wide applicative scope

Technology capitalizing on the versatile use of Tamoxifen-OH for controlling functions of multiple types of proteins.

Photochemical stability

Caged Cyclofen-OH liberates Cyclofen-OH, a highly photostable compound in contrast to Tamoxifen-OH encountering photodegradation under illumination.

Favorable wavelength ranges for uncaging

Uncaging requires either UV-A light or a strong IR laser. Visible light is inactive, which facilitates the experiments with biological samples.

Simple conditioning

Caged Cyclofen-OH is cell-permeant and can be added either in the external medium or directly injected for conditioning.

Excellent chemical stability

Caged Cyclofen-OH does not generate any basal activation of protein function and it benefits from an excellent temporal resolution upon uncaging.

Applications

Actiflash is a great R&D tool to convert your inducible ERT model into a photo-inducible one. It is very helpful if you want to control transcription (using Gal4-UAS) or induce recombination (using Cre-lox) in space and/or time for in-vivo cell tracking experiments and more.
Actiflash has been successfully used with various cell lines as well as zebrafish embryos. Actiflash will work best on zebrafish embryos from 12 to 48hpf.

Find below a plasmid list that can be used with Actiflash. All these plasmids are available from Addgene.

Technical Information

Lifetime: upon receipt, Actiflash is stable for years at 2-8°C. Once solubilized in a DMSO solution (typically at 10 mM), Actiflash can be stored at -20°C for several months. Always wrap the vials containing the aliquoted Actiflash with aluminium foil to protect it from light.

How to use Actiflash in brief:

Calibration of the Actiflash concentration

It is advised to first establish the extent of phenotype sought for as a function of the Tamoxifen-OH concentration. Then the concentration of Actiflash used for sample conditioning is fixed at Tamoxifen-OH concentration causing 100% of the desired phenotype (in general 3-5 μM in cultured cells and zebrafish embryos).

Conditioning with Actiflash

Incubate your samples in a serum-free medium for 90 mins, away from light.

Actiflash photoactivation

Illumination of Actiflash may be performed with UV (325-425nm range) light or multiphoton excitation (at 750 and 1064 nm with two- and three-photon excitation, respectively) to release Cyclofen-OH. You can use either benchtop UV lamps or light sources installed onmicroscopes.

The calibration of the photoactivation

The objective is to provide enough photons to exhaust the conversion of the Actiflash but without generating detrimental side-effects on the biological sample.Simply analyze the phenotype recovery with decreasing illumination duration. Then determine the shortest illumination duration leading to 100% uncaging of Actiflash.

What types of samples has Actiflash been tested on ?

Actiflash has been successfully used with various cell lines as well as animal models:
● Cell lines: CV1, mammalian epithelial cells (MDCK)
● Zebrafish: 12 hpf – 2 dpf embryos
● Mice
We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Will Actiflash work on zebrafish embryos older than 2 dpf?

Actiflash works optimally on embryos below 2 dpf when  following the provided protocol of use. On older embryos, protocol adjustments might need to be implemented to achieve a satisfactory photoconversion (i.e. heart injection, electroporation, etc).

Which Protein-ERT constructs have been previously photoactivated by Actiflash?

You will find below a non-exhaustive list of proteins that have been fused to the estrogen receptor:
● Recombinase Cre
● Tyrosine-protein kinase ABL1
● Cellular tumor antigen p53
● Proto-oncogene c-Fos
● Endothelial transcription factor GATA-2
● CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase4 (GAL4)

How should I store Actiflash?

Actiflash is provided as a powder. It is stable for years at 2-8°C. Once solubilized in a DMSO solution (typically at 10 mM), Actiflash can be stored at -20°C for several months.  Always wrap the vials containing the aliquoted Actiflash with aluminium foil to protect it from light.

How can I validate my protein-ERT construction?

You can perform experiments with Tamoxifen-OH as a positive control.

How can I validate photoactivation efficiency?

After conditioning your sample at a given concentration (e.g. 5 μM) with Actiflash,
illuminate your samples with the shortest illumination duration leading to the maximum
phenotype recovery. Compare with the results obtained with Tamoxifen-OH (1 μM).

Which plasmid should I use?

Any plasmid containing the ERT sequence should work.

Please find below a list of 52 plasmids developed by Sophie Vriz and Michel Volovitch in The Sophie Vriz Lab, who have the ownership of citations related to these plasmids. These 52 plasmids are all available on Addgene

Type Addgene Vriz lab name & link to Addgene Coding sequence Main use Regulatory sequences
ERT2 ready for fusion
184057 pCSnERT(#186) nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
ERT2 fused to CRE
184058 pCSCRET2(#323) 6myc-CRE-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184059 pT2EFCRET(#324) CRE-ERT2 Zebrafish Expression X.laevis-EF1
184060 pT2h70CRET(#325) CRE-ERT2 Zebrafish Expression D.rerio-hsp70, SP6
184061 pT22iU66CriT(#387) CRE-ERT2 Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi, SP6
184062 pT22iD36CriT(#388) CRE-ERT2 Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Dio3
ERT2 fused to Fluorescent Protein or Fluorogen-activated peptide
184063 pCSmChnERT(#99) mCherry-nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184064 pCSKaedenERT(#100) Kaede-nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184065 pCSGFPnERT(#205) eGFP-nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184066 pT22UbGfpnE4PCh(#301) eGFP-nls-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184067 pT2iC6Dronpa2nlsER4(#636) Dronpa2-2nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184068 pT2iC6FAST1nls2ER4(#638) YFAST-2nls-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
ERT2 fused to Gal4 activator
184069 pCSTG4FER3(#190) Gal4bdFF-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184070 pT22gCfpUbiG4FER4P2A(#249) Gal4bdFF-ERT2-P2A Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184071 pT2iaTubG4FER3(#940) Gal4bdFF-ERT2 Zebrafish Expression C.auratus-a1Tub
184072 pT22i2Shh24G4FER3(#1041) Gal4bdFF-ERT2 Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Shha+ABC
184073 pT26CfpSox10G4FER3(#1048) Gal4bdFF-ERT2 Zebrafish Expression, stable transgenesis (blue heart) D.rerio-Sox10
184074 pT26i4Gli8G4E3(#1254) Gal4bdFF-ERT2 Zebrafish Expression, stable transgenesis (blue heart) D.rerio-GliRE
ERT2 fused to various kinases, inhibitors, redox enzymes
184075 pCSZPKIaHAERT(#118) ZfPKAinhiba-HA-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184076 pCSZPKIamutHAERT(#119) ZfmutPKAinhiba-HA-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184077 pCSmp38ERT(#143) 5myc-ZfP38a-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184078 pT22U6CatalE4PCh(#767) CatalDeltaC-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi, SP6
ERT2 fused to various vertebrate transcription factors
184079 pCSmEn2ERT(#117) 6his-myc-chkEn2-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184080 pCSmEn2SRERT(#231) 6his-myc-chkEn2(SR)-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184081 pCSmEn25EERT(#232) 6his-myc-chkEn2(5E)-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184082 pCSmEn2n4ERT(#234) 6his-myc-chkEn2(n4)-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184083 pT22gCfpUbm5En2E4PCh(#251) 5myc-chkEn2-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184084 pT22UbHEn2E4PCh(#262) 3HA-chkEn2-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184085 pT22UbhaEng2aE4PCh(#271) 3HA-ZfEng2a-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184086 pT22UbhaEng2bE4PCh(#272) 3HA-ZfEng2b-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184087 pT22Ubm5Eng2aE4PCh(#283) 5myc-ZfEng2a-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184088 pT22Ubm5Eng2bE4PCh(#284) 5myc-ZfEng2b-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184089 pT22Ubm5En2SRE4PCh(#302) 5myc-chkEn2(SR)-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184090 pCSm5En2CER4(#457) 5myc-chkEn2(C>S)-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184091 pT22Ubm4Eng2bKRPC(#493) 4myc-ZfEng2b(WW>KK)-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184092 pT22gcfUbm5En25EE4PCh(#552) 5myc-chkEn2(5E)-ERT2-P2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184093 pCSm5En2DER4(#1062) 5myc-chkEn2(C>D)-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184094 pT22cUm5En2E4P3Che(#1188) 5myc-chkEn2-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184095 pT22cUm5En2SE4P3Che(#1189) 5myc-chkEn2(C>S)-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184096 pT22i5uasm5En2E4P3Che(#1208) 5myc-chkEn2-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) 5xUAS, SP6
184097 pT22i5uasm5En2SE4P3Che(#1209) 5myc-chkEn2(C>S)-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) 5xUAS, SP6
184098 pT22i5uasH3En2SE4P3Che(#1212) 3HA-chkEn2(C>S)-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) 5xUAS, SP6
184099 pT22i5uasm5En25EE4P3Che(#1213) 5myc-chkEn2(5E)-ERT2-5’fP2A-mCherry Zebrafish Expression, stable transgenesis (blue eye) 5xUAS, SP6
184100 pT2iD6Hoxa13aER4(#685) ZfHoxa13a-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184101 pT2iD6Hoxa13bER4(#686) ZfHoxa13b-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
184102 pT2iU6m5Hoxa13bE4PCh(#765) 5myc-ZfHoxa13b-ERT2-P2A-mCherry Zebrafish Expression D.rerio-Ubi, SP6
184103 pCSThaP6ERT(#184) HA-MmPax6-ERT2 Mammalian Expression, mRNA in vitro synthesis sCMV, SP6
ERT2 fused to Caspase-9 (apoptosis actuators and sensors)
184104 pT2iApowriter2(#336) myr-DIAP1-5myc-tEosFP-nls’-P2A-Casp9-ERT2 Zebrafish Expression D.rerio-Ubi
184105 pT22Apowriter2(#340) myr-DIAP1-5myc-tEosFP-nls’-P2A-Casp9-ERT2 Zebrafish Expression, stable transgenesis (blue eye) D.rerio-Ubi
184106 pT2iU6Apowriter2N(#355) myr-DIAP1-5myc-tEosFP-nls’-P2A-Casp9-ERT2 Zebrafish Expression D.rerio-Ubi, SP6
184107 pT22iuasvApoWtEos3N(#486) myr-DIAP1-2nls-tEosFP-nls’-P2A-Casp9-ERT2 Zebrafish Expression, stable transgenesis (blue eye) 4xUAS
184108 pT22iuasvApoWChe(#488) myr-DIAP1-nls’-mCherry-P2A-Casp9-ERT2 Zebrafish Expression, stable transgenesis (blue eye) 4xUAS

How can I determine the required duration of illumination that will activate Actiflash with my samples?

With the Actiflash conditioned samples, analyze the phenotype recovery observed using a
100% laser power and a decreasing illumination duration. You will thus determine the
shortest illumination duration leading to 100% uncaging.

Examples of the duration required for uncaging 100% of Actiflash:

• One-photon excitation at a 365 nm wavelength with a benchtop UV lamp having a power
range of 4-6 W (e.g. for global photoactivation): 5 minutes.
• One-photon excitation in the 350-405 nm, laser excitation power P = 10 μW focused on a
spot of diameter 10 μm: a few seconds.
• Two-photon excitation delivering 200 fs pulses at 750 nm and P = 10 mW in a cell of volume
V =100–1000 μm3: 1 second.

Stencell - Removable PDMS cell culture chambers

Ready-to-use silicon chambers to stick and remove for cellular confinement or dynamic assays, such as migration or wound-healing.

Each product contents 100 stencils (2 sheets of 50 stencils)

Ready-to-use silicon chambers to stick and remove for cellular confinement or dynamic assays, such as migration or wound-healing.

Each product contents 100 stencils (2 sheets of 50 stencils)

Description Catalog Number
Solo : 1 circular well – Diam. 12mm STU-STE-1WL
Quartet : 4 circular wells – Diam. 3mm STU-STE-4WL
Nonet : 9 circular wells – Diam. 3mm STU-STE-9WL
Allegro : 2 oblong wells spaced by 0.50 mm STU-STE-LRG
Presto : 2 oblong wells spaced by 0.25 mm STU-STE-THN

Stencell are ready-to-use silicon chambers that you can stick and remove for cellular confinement or dynamic assays, such as migration or wound-healing.
Stencell was designed to standardize your experiments and minimize the consumption of reagents.
It is very helpful when you are working with super expensive reagents or very rare cell lines, and when you want to test various experimental hypothesis.

Applications

Cell migration and wound healing with Presto and Allegro

  • Interaction dominant/dominated cells
  • Defined regions of Rab5A overexpression

Parallelized experiments with Quartet and Nonet

  • Differentiation of stem cells or zebrafish embryonic cells
  • Cell behavior in confined conditions

Confined experiments with Solo

  • Fixed epithelial cells growing confluent

Technical information

Material: PDMS
Kit content: 100 stencils (2 sheets of 50 identical stencils)
Sizes & shapes:
– Solo: 1 circular well – Diam. 12 mm
– Quartet: 4 circular wells – Diam. 3 mm
– Nonet: 9 circular wells – Diam. 3 mm
– Presto: 2 oblong wells spaced by 0.25 mm
– Allegro: 2 oblong wells spaced by 0.50 mm
Storage: up to 2 years, at room temperature under their protective sheets.

How to use Stencell:

Video protocol: Stencell for wound healing experiments

Find below advice & tips for Stencell use:

Stencell was designed by  Vincent Studer,  Pierre-Olivier Strale  and  Aurélien Pasturel.
They were hosted by the Cell Organ-izers joint research laboratory (CNRS-Alvéole).

Original publication:
Pasturel, A., Strale, P. O., & Studer, V. (2020). Tailoring Common Hydrogels into 3D Cell Culture Templates. Advanced healthcare materials9(18), e2000519. https://doi.org/10.1002/adhm.202000519

AgarSqueezer

The device to confine and study your cell behavior within a physiological rigidity range.

Kit contents:
. 1 Agarsqueezer device
. 2 needles

Size Catalog Number
1 device CRI-SOF-0
1 device + 1 microscope holder CRI-SOF-1
2 devices + 1 microscope holder CRI-SOF-2

Silicium wafers to mold confining pillars of various heights can be bought separately here.

A technology developed by Audrey Prunet, Gilles Simon, Hélène Delanoë-Ayari, Véronique Maguer-Satta & Charlotte Rivière – ILM Lyon, France

AgarSqueezer is a microscope slide chamber equipped with a molded agar-based compression system. Use it to apply an instant & homogeneous compression on your cells, and study their response to short and long-term confinement.

Physiological rigidity

Contrary to PDMS, the mechanical properties of agarose reproduce stiffness of the in vivo microenvironment (1-150 kPa).

Long-term confinement

As the porous nature of agarose facilitates nutrient and oxygen diffusion, the AgarSqueezer provides a safe environment for long-term cell culture. Keep your cells confined for up to 10 days!

Flexibility

By modulating confinement level, matrix stiffness & composition or coating with ECM proteins, AgarSqueezer leaves you plenty of options to reproduce environmental cues.

Applications

Agarsqueezer has been successfully used to confine adherent and non-adherent cells, including:
> Human: primary T-lymphocytes, TF1 & ML2 leukemic cells, HS27A fibroblasts, MCF10A breast cells, MDA-MB-231 breast cancer cells, U-2 OS osteosarcoma cells, PC-3 & DU 145 prostate cancer cells, HT29 & HCT116 colorectal adenocarcinoma cells and HT1080 fibrosarcoma cells
> Murine: megakaryocytes, osteocyte-like cells MLO-Y4, primary muscle cells & primary dendritic cells
> Plant: cells from Arabidopsis roots
> 3D cell cultures: mice gastruloïds

 

Compatible assays:
> Live imaging*: cell viability, proliferation, migration, morphology, nuclear deformability, cytoskeleton reorganization, cell tracking, etc
> Standard molecular analysis (immunostaining, western blot, qPCR, flow cytometry, etc): changes in gene/protein expression, proliferation, morphological analyses, etc

*Cells can be added with fluorescent probes or stimulated with drugs during confinement.

 

Imaging modes:
Inverted microscopes equipped with a standard adjustable petri dish holder, with any imaging mode: phase-contrast, epifluorescence, confocal, etc.

Kit contents

1x Agarsqueezer compression device
1x 16G flat cut needle to make holes in the agar gel, facilitating diffusion of culture medium or drugs during the experiment
1x 20G flat cut needle (same as above)
(Optional) 1x microscope stage insert (102.5mmx143.5mm), holding up to 2 AgarSqueezers in live imaging chambers (Okolab or equivalent).
In addition to basic kit contents, we recommend using a silicium wafer to mold your confining pillars in agarose. We provide four different wafers to mold pillars of various heights. Choose among our four pillar heights available depending on your desired application and add silicium wafer(s) to your order here.

Compression of immature TF1-GFP hematopoietic cells

Quantification of cell morphology under confinement. (A–C): Morphology of immature TF1-GFP hematopoietic cells for control (A) and for 30 μm and 5 μm (B and C, respectively). Scale bar = 20 μm. (D) Quantification of projected area by automatic image analysis. Projected area was similar for control and 30 μm, while it significantly increased for 5 μm confinement (unpaired t-test n = 160 at least for each condition). (E and F): z-Section of unconfined (E) and confined (F) cells. Scale bar = 10 μm.
et al. Lab on Chip, 2020.

Human primary T-lymphocytes migrating in confined conditions

Human T-lymphocytes isolated from blood were seeded in the Agarsqueezer and confined under 5µm pillars. Images were taken every 10sec for 16.5min using an inverted Zeiss Z1 automated microscope (10X objective).

Credits: Marie-Pierre Valignat – Adhesion & inflammation lab, Aix-Marseille University, France

Arabidopsis root cells confined using Agarsqueezer

Arabidopsis thaliana Col-0 root cells stained with Calcofluor (cell wall) and imaged with a confocal microscope either in a traditional culture setting (left), or after 24h of confinement in the Agarsqueezer using the 30µm (middle) or 5µm (right) pillars.

Credits: Léa Bogdziewiez – UPSC – Sveriges lantbruksuniversitet, Sweden

Compression of mouse primary myoblasts using AgarSqueezer

C57 primary myoblasts stained with Hoechst were imaged in the AgarSqueezer before compression (left panel), and after 1.5h of compression under 2.5 µm height pillars (middle & right panels).
Credits: Dr. Hind Zahr & Dr. Alice Varlet, Lammerding Lab – Meinig School of Biomedical Engineering, Cornell University, United States

Observation of vesicles at septum during cell division of mutant E. coli (W3110 tolR – Palmcherry),
in LB 1/2 medium, using Chitozen.
Set-up mimicking the complexity of the tumor microenvironment

How to use Agarsqueezer:

Video protocol: How to assemble and use Agarsqueezer device

Read the full protocol

Encountering issues with Agarsqueezer? Check out our

How to use AgarSqueezer in pictures

Which types of cells has Agarsqueezer been tested for?

  • The Agarsqueezer device has been successfully used to study compression of adherent and
    non-adherent cells, including:

    Human cells:
    Fibrosarcoma (HT-1080)
    Osteosarcoma (U-2 OS)
    Colorectal adenocarcinoma
    (HT29 & HCT116)
    Prostate cancer (PC-3 & DU 145)
    Breast cancer (MDA-MB-231)
    Leukemia (TF1 & ML2)
    Megakaryocytes
    Fibroblasts (HS27A)
    Breast cells (MCF10A)
    Primary T-lymphocytes

    Murine cells:
    Osteocyte-like cells (MLO-Y4)
    Primary dendritic cells
    Primary muscle cells

    Plant cells:
    Arabidopsis Thaliana root cells

    3D cell culture:
    Mice gastruloids

What are the benefits of using an agarose-based instead of a PDMS-based cell confiner?

  • Physiological relevance: the agarose hydrogel rigidity is tunable and reproduces the
    stiffness of the in vivo microenvironment (1-150 kPa), contrary to PDMS which rigidity
    is several orders of magnitude larger (MPa).
  • No reduction of the available drug dose: agarose hydrogels overcome drug adsorption
    issues associated with PDMS.
  • Passive medium renewal & oxygen diffusion: the porous nature of agarose hydrogels
    allows free diffusion of oxygen, salts and small molecules and therefore minimize
    nutrient depletion, allowing for long-term confinement studies.

Which types of analyses can I carry out with Agarsqueezer?

The system is fully compatible with live imaging and time-lapse microscopy and all
immunostaining steps can be performed in situ. Alternatively, cells can be collected for
classical biochemical and molecular analysis (qPCR, Western Blot, flow cytometry, etc).

How long can cells be cultured in the Agarsqueezer for?

The device has been designed for long-term confinement studies and the porous nature of
agarose enables passive medium renewal as well as oxygen diffusion. You can safely grow cells
up to 10 days in the AgarSqueezer device.

Can I stimulate my cells with drugs or other compounds during confinement studies?

Yes, the use of agarose allows free diffusion of small molecules (size <30 nm in 2% agarose).
You can easily add drugs, antibodies or other compounds at any point during cell confinement
studies thanks to an open access to the reservoir. Drug availability and activity within the
system has been validated using in situ addition of the tyrosine kinase inhibitor imatinib, and
diffusion experiments showed that 3 hours are required for the diffusion of small molecules
such as BSA or FITC.

Which pillar heights are available, and which one should I choose?

The currently available pillar heights are:
– 2.5μm for inducing highly confined conditions
– 5μm for inducing moderately confined conditions
– 30μm to serve as an unconfined control
– 100μm to confine 3D cellular structures or immobilize them for imaging

Those dimensions have been determined based on the size of most classical human cell lines.
The resulting level of compression applied will depend on your sample type, size and the use
of mono- or multi-cell layers.

How can I tune the matrix stiffness?

You can easily modulate the matrix stiffness by tuning the type and concentration of agarose used to mold the pillars. Using a standard agarose at the recommended 2% concentration will result in a storage modulus within the range of 50-200kPa. To accentuate the matrix stiffness and the resulting confinement applied, the agarose concentration can be increased. For example, a 3% agarose was found to be more efficient at confining cell cultures from Arabidopsis roots. If you wish to decrease the matrix stiffness, we recommend using a “low
melting point” or “ultra-low gelling” temperature agarose to yield a storage modulus in the range of ~1kPa. For a similar stiffness on top and bottom walls, the coverslip can be coated with an additional soft agarose layer.

How can I tune the matrix composition?

To analyze the role of various ECM proteins on cell response to mechanical confinement, you
can coat the coverslip with adhesive proteins (fibronectin, collagen, Matrigel, etc). In addition,
the agarose can be added with collagen, PEGDA with covalently immobilized RGD peptides or
silk.

What is included in the AgarSqueezer kit? Is there anything I need that is not provided?

Our AgarSqueezer kits contain all elements making up the compression device, a wafer to mold your agarose gel and some needles specifically designed to form your entry ports in the agarose. Optionally, an insert (102.5mmx143.5mm)​ designed to image 2 AgarSqueezer devices in parallel can be added to your order. This insert will fit into Okolab imaging chambers or equivalents.

All you need to have on your side is:
• Agarose powder
• Distilled water
• Round 22 or 30 mm coverslips
• A screwdriver
• For imaging: a standard petri dish holder can be used to place and image the AgarSqueezer on inverted microscopes. Use objectives up to 40X for optimal performance. Although objectives of 60X and above can be used, they will result in a shorter observation area and we cannot guarantee an optimal working distance depending on your microscope.

Can I re-use the AgarSqueezer device?

Yes, the AgarSqueezer device can be re-used as long as it is thoroughly sterilized by autoclaving
before each experiment. Simply keep in mind that the agarose solution should be prepared
no more than one day before your experiment.

Agarsqueezer has been designed by Audrey Prunet, Gilles Simon, Hélène Delanoë-Ayari, Véronique Maguer-Satta and Charlotte Rivière (ILM Lyon, France).

Original publication:
Prunet, A., Lefort, S., Delanoë-Ayari, H., Laperrousaz, B., Simon, G., Barentin, C., Saci, S., Argoul, F., Guyot, B., Rieu, J. P., Gobert, S., Maguer-Satta, V., & Rivière, C. (2020). A new agarose-based microsystem to investigate cell response to prolonged confinement. Lab on a chip20(21), 4016–4030. https://doi.org/10.1039/d0lc00732c

Additional publications:

Mitosis down-regulates nuclear volume and resets nuclear envelope folding of cancer cells under prolonged confinement
Malèke Mouelhi, Alexis Saffon,  Hélène Delanoë-Ayari, Sylvain Monnier, Charlotte Rivière, Biorxiv 2023
doi: https://doi.org/10.1101/2023.05.11.540326

Mitosis sets nuclear homeostasis of cancer cells under confinement
Maleke Mouelhi, Alexis Saffon, Morgane Roinard,  Helene Delanoe-Ayari,  Sylvain Monnier,  Charlotte Riviere, Biorxiv 2023. doi:  https://doi.org/10.1101/2023.05.11.540326

Etched silicium chip for molding AgarSqueezer pillars available patterns: pillars of 2.5 – 5 – 30 – 100 µm height

Size Catalog Number
5 µm P073-5
2,5 µm P073-2.5
30 µm P073-30
100 µm P073-100

Agarsqueezer Silicium wafer chip is a companion product of Agarsqueezer.

Stampwell

A stamp to mold agarose wells to culture and image spheroids and organoids

Size Catalog Number
V300 GRE-MOU-V300
V500 GRE-MOU-V500

Stampwell stamps imprint arrays of wells in hydrogels in just one time. Nothing more to do than load your samples into the wells and image. The Stampwell shapes have been optimized for the imaging of spheroids and organoids.

V Shape 300µ:
. Number of pins: 42
. Depth of the well: 1 mm
. Well bottom diameter: 300µm – Shape: circular
. Well upper side: 1mm*0.5mm – Shape: rectangular

V Shape 500µ:
. Number of pins: 42
. Shape of the pins: V
. Depth of the well: 1 mm
. Well bottom diameter: 500µm – Shape: circular
. Well upper side: 1mm*0.5mm – Shape: rectangular

Imprinted V-shape well in agarose, thanks to Stampwell V-300

Soaked in fluorescein, negative rendering

Image credit: Gaëlle Recher – Bordeaux

hiPSCs cyst in alginate capsule in a V-shape well made with Stampwell

scale bar: 500µm

Image credit: Gaëlle Recher – Bordeaux

Cell-filled alginate capsule in the V-shape well

Notice that the focus moving through the well height is easily visible (lines in focus Vs out of focus)

Image credit: Gaëlle Recher – Bordeaux

V-shape imprinted wells

Filled with a single cell-loaded alginate capsule: corresponding imaged with a stereomicroscope and stitched. Gel pad is made of 2% agarose. (Scale bar: 2mm)

Image credit: Gaëlle Recher – Bordeaux

Apical-out 10-day human airway organoids imaged in a V-shape Stampwell in comparison with suspension plates

Airway organoids were generated from a human bronchial cell line in hydrogel and transferred either to microwells made using a V-shape Stampwell, or suspension plates, for turning them inside out and imaging.
Credits: Signe Lolle – Danmarks Tekniske Universitet, Denmark

How to use Stampwell:

 

Read the full protocol:

Video protocol: How to use Stampwell

Protocol overview

1. Pour liquid agarose (or other hydrogel)

2. Place the stamp

3. Reticulate the hydrogel

4. Remove the stampv

Find below advice & tips for Stampwell use:

Mold agarose wells to position and image zebrafish larvae and embryos

Size Catalog Number
Embryo 1 GRE-MOU-REC
Embryo 2 GRE-MOU-LAI
Larvae 1 GRE-MOU-DEV
Larvae 2 GRE-MOU-ADU

Stampwell stamps imprint arrays of wells in hydrogels in just one time. Nothing more to do than load your samples into the wells and image. The Stampwell shapes have been optimized for the imaging of fish embryos or larvae up to 20 dpf.

Save time: no need to manually position your samples individually. Fish embryos/larvae will self-position in the wells.

Boost your assay reproducibility: once loaded in the wells, your samples are perfectly positioned, aligned and located on a similar focal plane.

Open system: direct access to embryos/larvae for adding external compounds (i.e. drug screening).

4 different shapes
depending on the size and orientation of your fish embryos.

Embryo 1 (ie Rectangular) 
. Number of pins: 35
. Shape of the pins: rectangular
. Length * width of the wells: 2 mm * 0,65 mm

Embryo 2: ​
. Number of pins: 18
. Shape of the pins: a drop
. Length of the wells: 3.90 mm
. Largest width of the wells: 0.88 mm

Larvae 1: ​
. Number of pins: 5 large pins ideal for the 9-14 dpf zebrafishes + 5 medium pins ideal for the 6-9 dpf zebrafishes + 5 small pins ideal for the 3-6 dpf zebrafishes
. Shape of the pins: fish body and tail
. Length of the 5 large wells: 6 mm, 2.7 for the body and 3.3 for the tail
. Length of the 5 medium wells: 5 mm, 2.25 for the body and 2.75 for the tail
. Length of the 5 small wells: 4 mm, 1.8 for the body and 2.2 for the tail

Larvae 2: ​
. Number of pins: 5 large wells ideal for the 15-20 dpf zebrafish or 39-42 dpf medaka larvae + 5 small wells ideal for the 1-2 dpf zebrafish
. Shape of the pins: prism
. Length of the 5 large wells: 10 mm
. Length of the 5 small wells: 3 mm
. Well depth: 1 mm

Parallelized imaging of zebrafish embryos using the Embryo 1 Stampwell

48hpf embryos (anti-HuC/D, ab210554, abcam)

Credits: Matthieu Simion, CNRS, 2022

Improved positioning of 2dpf zebrafish larvae using the Embryo 2 Stampwell

Credits: Dr Rui Monteiro – Institute of Cancer and Genomic Sciences, University of Birmingham, United Kingdom

Zebrafish embryos imaging in agarose wells made with Stampwell Embryo 1

Bar: 1mm (projection of Z-stack – confocal microscope)
Credits: Gaëlle Recher – Bordeaux 2019

Heart imaging in a Larvae 1 Stampwell

Top picture: green-heart 3dpf zebrafish larvae placed in ventral view. Middle & bottom pictures: 3dpf zebrafish larvae PFA-fixed,
mounted in glycerol and inserted in Larvae 1 Stampwell (anterior to the left). Pictures were taken using a Leica Spectral Confocal SP5.
Credits: Dr. Giovanni Risato, Prof. Natascia Tiso and Alina Ramazanova – Department of Biology, University of Padova, Italy

Live imaging of beating hearts in early zebrafish embryos using the Larvae 1 Stampwell

Anesthetized 3 dpf embryos expressing a fluorescent biosensor in the heart were mounted in agarose microwells made using the Larvae 1 Stampwell and imaged in ventral view.
Credits: Prof. Juan Llopis – CRIB, Universidad de Vastilla La Mancha, Spai

How to use Stampwell:

 

Read the full protocol:

Video protocol: How to use Stampwell

Protocol overview

1. Pour liquid agarose (or other hydrogel)

2. Place the stamp

3. Reticulate the hydrogel

4. Remove the stampv

Find below advice & tips for Stampwell use:

SensiDeath - Ultra-sensitive human cell death assay

SensiDeath is a novel sensitive kit for human cell death detection. Taking benefit from qPCR high sensitivity, SensiDeath provides a simple method to detect cell death at its earliest stages by quantifying genomic DNA fragments released in the cytoplasm. Use it to get reliable measurements of apoptosis across a range of cell populations.

Products

Product Catalog No.
SensiDeath – Ultra-sensitive human cell death assay -300 assays IDY-SD-300-IDY
SensiDeath – Ultra-sensitive human cell death assay – 1000 assays IDY-SD-1000-IDY

Fast & simple
As simple as a qPCR. Lyse your cells, collect the cytosolic fraction and get results in less than 2h !

Extended dynamic range
Get reliable data and make sure to never miss out subtle changes in cell death.

Scalable
Compatible with 96-well plate format and efficient on small cell populations, SensiDeath is a convenient method for high-throughput cytotoxicity assays.

Early detection
Based on extremely sensitive cytosolic detection of genomic DNA, SensiDeath detects cell death as soon as it is initiated and up to its late phase.

How does SensiDeath work?

SensiDeath is based on a patented technology relying on the quantitation of genomic DNA released in the cell cytosol, a key process initiated upon cell death and a valuable marker of apoptosis. By amplifying genomic DNA present in isolated cytosolic fractions, you can quickly compare relative levels of apoptotic cells across a wide range of samples.

  1. Lyse your sample using our special lysis buffer disrupting cell plasma membrane specifically
  2. Centrifuge your lysate to recover the cytosolic fraction
  3. Amplify specific sequences present in the genome by quantitative PCR using our optimized primers
  4. Assess cell death that is directly proportional to cytosolic enrichment in genomic DNA

Applications of SensiDeath

Outputs: comparative measurements of cell death levels for cytotoxic or viability assays. SensiDeath can be used as a reliable method to distinguish changes in apoptosis*.
*Check out our Results section for detailed results.

Sample types: human cells (adherent & suspension)*.
*Check out our FAQ section for a full list of validated cell lines.

Analysis: PCR-based techniques (quantitative PCR, digital droplet PCR).

Kit contents of SensiDeath

All our kits include a pair of optimized primers designed to amplify a genomic repeat DNA element. They have been thoroughly selected and optimized to combine high sensitivity, specificity and reproducibility in cell death detection.

Our kits also include a concentrated lysis buffer, developed and optimized to recover cytosolic fractions by disrupting the cell plasma membrane specifically while maintaining integrity of the nuclear membrane as well as that of other cellular organelles.

Small kit (>300 assays*)
Primer mix (20X): 300 µL

SensiDeath Lysis buffer (6X): 25 mL

Extended kit (>1000 assays*)
Primer mix (20X): 1 mL

SensiDeath Lysis buffer (6X): 85 mL

*Number of assays are given for 20 µL qPCR reactions. More assays can be carried out with each kit if working with smaller qPCR reaction volumes.

 

Storage: the 6X lysis buffer can be stored at 4°C for at least 1 year. Primers can be stored at 4°C for one month, or at -20°C for long-term storage.

SensiDeath provides increased sensitivity in cell death detection compared to existing methods

Caspase-Glo® 3/7 or SensiDeath kits were used to measure cell death in increasing quantities of MOLM14 cells treated with 1 µM Etoposide for 16 hours. SensiDeath provides a 4-fold increase in detected signal compared to Caspase-Glo® 3/7 in treated samples and shows a lower detection limit.
Image credits: Equipe ANUBIS, Université de Toulouse, France

Extended dynamic range of SensiDeath compared to existing methods

Annexin-V or SensiDeath methods were used to measure cell death in MOLM14 cells treated with increasing concentrations of Etoposide for 16 hours. Compared to Annexin-V, with which a signal upper limit is reached from 2.5 µM Etoposide, SensiDeath provides an extended dynamic range with additional intermediate values available up to 10µM Etoposide.
Image credits: Equipe ANUBIS, Université de Toulouse, France

How is cell death detected ?

During cell death, fragmented genomic DNA accumulates in the cytosol. This process takes place as soon as cell death is initiated, and persists up till later stages. Using a selective lysis buffer and qPCR primers, SensiDeath allows a rapid and highly sensitive cell death quantification by detecting genomic DNA fragments released in the cytosol.

Can SensiDeath be used for apoptosis measurement ?

Yes, the specific primers provided in the SensiDeath kit are designed to amplify apoptotic DNA generated as soon as cell death is initiated. Several assays confirmed that SensiDeath allowed to distinguish changes in apoptosis using a variety of apoptotic inducers. Check out our Results section for detailed results.

Which stage(s) of apoptosis are detected by SensiDeath?

The presence of nuclear DNA in the cytosol can be detected in early, mid and late phase apoptosis. The cytosolic concentration of nuclear DNA will be maximal in the mid-phase, but the extreme sensitivity of the SensiDeath method enables its detection in its earliest phases too.

How is cell death quantified?

The SensiDeath method relies on the amplification by qPCR of genomic DNA present in the cell cytosolic fraction. By amplifying repeated genomic sequences present in the cytosolic fraction, you can assess the relative cytosolic enrichment in genomic DNA in comparison to an untreated, negative control. This enrichment will be directly proportional to the cell death levels in your sample.

Which samples can be analyzed with SensiDeath?

SensiDeath has been successfully used to detect cell death in a variety of human cell lines, including:
– Adherent cells: HepG2, MDA-MB-231, HeLa, SK-Hep, HEK293, HUVEC, primary fibroblasts, etc
– Suspension cells: OCI-AML3, MOLM14, MV4-11, HL-60, THP-1, U937, etc

Please note that this list is non-exhaustive and SensiDeath could be used to assess cell death in any other human cell types.

Is SensiDeath compatible with high-throughput assays?

SensiDeath is perfectly well-suited for high-throughput and automated assay formats. Extracted cytosolic fractions can be analysed in 96-well or smaller plate formats. The centrifugation step can be replaced by a magnetic-bead system to purify cytosolic DNA fragments.

What genomic sequence in amplified?

We provide optimized primers for amplifying a 153 bp genomic repeated element. This primer pair has been meticulously selected to ensure high sensitivity, specificity and reproducibility.

How can SensiDeath distinguish between cell death and mitosis?

Indeed, some DNA can also be present in the cell cytosol during other processes, especially mitosis. Some tests have been carried out to check whether changes in the cell cycle phases might skew the obtained cytotoxicity data. Non-apoptotic cells were labelled with propidium iodide, and sorted using flow cytometry according to their cell cycle phase (G0/G1, S or G2/M). The SensiDeath protocol has been performed on these samples, and results showed similar qPCR signals across the different samples demonstrating that changes in the cell cycle phase did not affect the obtained data.

Is there any specific material or equipment required?

The SensiDeath kits contain a concentrated lysis buffer and optimized primers for genomic DNA amplification. The only reagent needed is nuclease-free water and any SYBR Green qPCR mix. In terms of equipment, you need a simple benchtop centrifuge for sample preparation and a thermocycler for performing the qPCR. You do not need any sophisticated equipment.

Is there a risk to recover nucleus content when collecting the cytosolic fraction?

After lysate centrifugation, nuclear fractions are pelleted at the bottom of the tube. A few microliters of the supernatant are collected for analysis, thus excluding the risk of nuclear contamination.

Can I store my samples before performing qPCR analyses?

Yes, nucleases are efficiently inhibited by the lysis buffer. Extracted cytosolic fractions can be kept at -20°C for months before being assayed. This is particularly useful when performing kinetics studies, or simply to save time by assaying multiple samples in parallel.

Can SensiDeath lysis buffer be added directly to culture medium?

Yes, the lysis buffer can be added directly to cell culture medium as long as its final concentration is 1X.

Is it normal to obtain a qPCR amplification signal in negative control samples?

Yes, the qPCR amplification signal will reflect the basal level of apoptosis in the control cell population.

Can mitochondrial DNA be present in the cytosolic fraction collected?

No, our lysis buffer provided in the kit has been specifically optimized to disrupt the plasma cell membrane while leaving the nuclear membrane, as well as that of all other organelles, perfectly intact. Therefore, there is no risk of retrieving any mitochondrial DNA in the isolated cytosolic fractions.

Can I use my own lysis buffer instead of that provided in the kit ?

The SensiDeath lysis buffer provided in the kit has been specifically developed for destructing the cell plasma membrane while keeping the membrane of nuclei and other organelles intact, therefore allowing the specific retrieval of the cytosolic fraction. In addition, its components have been optimized not to interfere with subsequent qPCR reactions. Therefore, we do not recommend using other lysis buffers as we cannot guarantee the accuracy of the obtained cell death measurements.

Can I recover nucleus and organelle contents after extracting the cytosolic fraction?

Yes, the SensiDeath lysis protocol preserves the integrity of the nucleus and other organelles (mitochondria, ER and Golgi) while extracting the cytosolic fraction. Therefore, it is entirely possible to recover the organelle and nucleoplasm contents to carry out complementary assays from the same lysate. This has been successfully done using detergent-based sequential subcellular fractionation for Western blot analysis.

GlowMito - Red fluorescent mitochondrial tracker

Red fluorescent probe labeling all mitochondria for live imaging & flow cytometry

GlowMito provides a non-toxic solution to quickly visualize the entire mitochondrial network in live samples.

GlowMito quickly penetrates cells and produces a bright & stable red labeling of mitochondria without inducing cell toxicity or altering mitochondrial functions. Simply add it to your culture medium, and image repeatedly for up to 3 days without washing!

Soluble in water and compatible with 2-photon microscopy, GlowMito is the solution of choice for thick tissues and in vivo experiments.

GlowMito is retained in the mitochondria of live samples following loss of membrane potential.

 

Technical Information about GlowMito

Colour: Red/near-infrared
Detection method : Fluorescence, one & two-photon excitation
Peak excitation/emission wavelengths: Ex:542nm/Em:690nm
Two-photon absorption peak: 790nm
Two-photon cross-section: 224 GM
Form : Liquid (resuspended in water)
Sub-cellular localization: mitochondria
For use with: epifluorescence & confocal microscopes, flow cytometers
Fixable: no

Kit contents of GlowMito:

1 vial contains 125 µL of an aqueous solution with a concentration of 1 mM for a final volume of 250 mL of imaging medium
Storage: Store at 4°C for up to 7 months. For long-term storage, aliquot and store at -20°C.

 

Applications of GlowMito:

Experimental outputs: live imaging & flow cytometry

GlowMito produces a bright red labeling of the entire mitochondrial network, including those with reduced potential. It is primarily intended to visualize mitochondria based on their presence rather than assessing their electrochemical properties. GlowMito does not need to be washed out: mitochondrial fluorescence will remain stable for up to 3 days without causing toxicity and can therefore be integrated in your long-term experiments.

GlowMito is perfectly suitable for studying mitochondrial morphology, movement (velocity, localization), tracking, dynamics (fusion/fission), biogenesis (replication & growth), autophagy, etc.

GlowMito can be used in combination with other probes (GFP, potentiometric dyes, calcium signaling probes, etc).

We do not recommend its use for the measure of mitochondrial mass or volume density.

Tested and validated on cell lines, tissues & unicellular organisms:

Humans: HEK293, HeLa, MCF-7, MDA-MB-231, HMLE, UACC-62, U2-OS, Gli36, HAEC, SH-SY5Y, A-172, A549, patient-derived skeletal muscle cells, primary smooth muscle cells & iPSC-derived cardiomyocytes, hiPSC-derived heart tissues.
Mice: primary cortical neurons, acute brain slices, isolated pressurized blood vessels
Monkey: COS-7
Parasitic protists: Trichomonas Vaginalis (hydrogenosome labeling)

GlowMito showed no internalization in yeast cells, Entamoeba histolytica, Giardia intestinalis parasites and seedlings.

Smooth muscle cell mitochondria labeling in an intact pressurized cerebral artery using GlowMito

This confocal microscope image depicts the smooth muscle cell layer of an intact pressurized cerebral artery, illustrating mitochondria (shown in magenta) along with the nuclei (shown in blue). Smooth muscle cell nuclei are elongated and run perpendicular to the vessel axis. Small, rounded nuclei correspond to adventitial cells on the vessel wall.
Credits: Image captured by Safa in Dr. Charles Norton’s laboratory – University of Missouri (US)

Axonal mitochondrial labeling in acute mice brain slices using GlowMito

300 µm thick acute mice brain slices from the primary visual cortex were labeled with 1 µM GlowMito and imaged on a 2-photon microscope (820 nm, 23 mW). Z-stack pictures were taken from top to bottom of the slice (2 µm steps between each image).
Credits: Lauren Panzera (Postdoctoral Fellow Higley Lab) & Michael Higley (Dept. Of Neurosciences) – Yale University, New Haven, CT USA

GlowMito produces a stable & bright labeling of mitochondria for 4 hours

GlowMito was directly added at 500 nM in live COS-7 culture medium. Cells were imaged repeatedly

 

Internalization & stability of GlowMito compared to a commercial mitochondrial probe

HEK293 cells were observed before and after 2, 4, and 6 minutes of incubation with GlowMito 500 nM.
Acquisition parameters: laser 2%, gain 15
A maximum of internalization is observed after 4 minutes.
Credits: Morgane LeMao, Guy Lenaers, Olivier Siri – MitoLab, Unité MitoVasc, Université d’Angers, CNRS UMR6015, Inserm U1083, Université de Marseille, CINAM UMR 7325 – 2023

 

Mitochondrial labeling in Aspergillus Fumigatus

10x10e3 spores were incubated for 30 min with 500nM GlowMito in YPD medium.

Credits: Géraldine Leman (Infections Respiratoires Fongiques IRF), & Rodolphe Perrot (Service Commun d’Imageries et d’Analyses Microscopiques SCIAM) – Université d’Angers, France – 2024

Mitochondrial labeling in iPS-derived cardiomyocytes

How to use GlowMito:

General enquiries

Where does GlowMito come from?

GlowMito was derived from a triamino-phenazinium belonging to the induline family. With their attractive absorption properties and excellent stability, these molecules have a long history as dyestuffs and attracted major attention in applications from photoreceptor or electrophotography agents to ingredient in color industry.

These derivatives also showed great potential to serve as new mitochondrial probes, although biological applications had so far been neglected. In particular, their appealing photophysical properties characterized by high fluorescence quantum yield and absorption cross-section make them ideal candidates for two-photon excitation microscopy. After having modified some of their substituents to make them internalized by cells and mitochondria more efficiently, GlowMito was born!

The newly synthesized compound, soluble in water, proved very efficient at quickly accumulating within mitochondria without inducing any cytotoxic effects or altering normal mitochondrial functions. Since then, GlowMito expanded the scope of mitochondrial probes by providing a solution of choice to label the entire mitochondrial network quickly and safely in live cells, deep tissues, and even anaerobic organisms.

What do we know of GlowMito labeling mechanism?

GlowMito is a cell-permeant probe that is rapidly internalized by cells and specifically target the mitochondria. The quick GlowMito penetration in cells was made possible by the introduction of lipophilic substituents in the original molecule, coupled with its cationic charge, thereby enhancing passive diffusion across cell membranes as it is observed for various lipophilic cations.

As known for various other lipophilic cations, GlowMito accumuation within mitochondria is at least partly driven by membrane potential. Yet, although the precise mechanisms are not yet unraveled, it is likely that GlowMito also relies on other membrane potential-independent mechanisms such as specific interactions with anionic species within the mitochondrial matrix, enhancing the retention of the dye within the mitochondria even with reduced potential.

How was mitochondrial specificity of GlowMito verified?

The strong ability of GlowMito to specifically target mitochondria has been meticulously checked by co-localization studies. More generally, the ability of lipophilic cations to specifically target mitochondria is already well-established and conjugating molecules to lipophilic cations is a commonly used method to develop mitochondria-targeted compounds.

Is GlowMito dependent on mitochondrial membrane potential?

Contrary to classical potential-dependent probes, GlowMito is minimally affected by membrane potential loss and will still produce a visible signal in mitochondria with poor membrane potential. This was demonstrated by pilot studies, later confirmed by early users, showing that GlowMito signal was higher than that of TMRM in presence of an uncoupler. One of our early users even found that GlowMito was able to label hydrogenosomes in the anaerobic organism Trichomonas vaginalis, that have only little -if any- membrane potential, and for which other probes like JC-1, DioC6 and TMRE were not working.

Which assays can GlowMito be used for?

GlowMito produces a bright, red labeling of the entire mitochondrial network, including those with reduced potential. It is perfectly suitable for:

· Live imaging: study of mitochondrial dynamics (velocity, localization), structural changes, multiplexing (potentiometric dyes, calcium signaling probes, etc)

· Downstream analysis: flow cytometry, oxygraphy, etc

We do not recommend its use to measure mitochondrial mass or volume density.

Can GlowMito be used to label fixed cells?

No, GlowMito can be used to label mitochondria in live samples only and is not retained after aldehyde fixation.

Which types of samples has GlowMito been used with?

So far, GlowMito has been successfully used to label mitochondria in the following biological samples:

· Human cells: HEK293, HeLa, MCF-7, MDA-MB-231, HMLE, UACC-62, U2-OS, Gli36, HAEC, SH-SY5Y, A-172, A549, patient-derived skeletal muscle cells, primary smooth muscle cells & iPSC-derived cardiomyocytes

· Monkey cells: COS-7

· Mice cells: primary cortical neurons

· Tissues: hiPSC-derived heart tissues & mice isolated pressurized blood vessels

· Parasitic protists: Trichomonas Vaginalis

GlowMito showed no internalization in yeast cells

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Protocol of use

Are there any reagents I need that are not provided?

GlowMito comes as a 1 mM stock solution in water, that you can directly dilute in your culture medium. All you need to have on your side is a culture vessel, pre-heated culture medium or your choice and your biological sample.

Which lasers/filters do I need to image GlowMito or detect it in flow cytometry?

The full excitation (solid line) and emission (dotted line) spectra of GlowMito in one-photon microscopy is presented below (Ex:542nm/Em:690nm):

For use in flow cytometry, we recommend using a 488nm laser and a 655-730 nm emission filter.

The absorption spectrum of GlowMito in two-photon microscopy is presented below (red line, cross-section = 224GM at 790nm):

How long does it take to label mitochondria with GlowMito?

The total procedure to label mitochondria in live cells should take no more than 40 min. For deep tissues, total labeling time might need to be extended up to 1.5 h.

How long is GlowMito signal stable for?

The GlowMito signal has been shown to remain stable & specific for up to 3 days with no sign of cell toxicity.

How long can GlowMito be stored for?

The 1 mM GlowMito stock solution can be stored at 4°C for one month. For long-term storage, aliquot to avoid repeated freeze/thaw cycles and store at -20°C for up to 6 months. Always store GlowMito protected from light.

How many experiments can be performed with one vial of GlowMito?

One vial contains 125µL of a 2,000X aqueous solution for a final volume of 250mL of imaging medium when used at the recommended working concentration. The optimal GlowMito working concentration might need to be increased up to 2-fold when working with deep tissues.

Troubleshooting

I am experiencing photobleaching when imaging with GlowMito

GlowMito can be prone to photobleaching when exposed to high-intensity and/or prolonged illumination in one-photon confocal microscopy. Keep in mind that GlowMito photobleaching should be minimal when imaged in 2-photon microscopy.

Here are a few tips you can implement to reduce GlowMito photobleaching:

· Adjust laser intensity and keep it to a minimum.

· Reduce exposure time and try to increase intervals between acquisitions where possible.

Note: capturing a series of short exposure images at regular intervals, instead of a single long exposure, can be used to assess mitochondrial dynamics over time.

· If not sufficient, using anti-fading agents will reduce photobleaching during prolonged imaging sessions.

Note: the ProLong Live antifade reagent has been successfully used in combination with GlowMito and enabled strong conservation of GlowMito signal after 15 sec of continuous light exposure on a confocal microscope.

ColorFlux - A visual indicator of bacterial efflux

Fast, reliable and non-toxic solution for measuring bacterial efflux.

ColorFlux is a fast, reliable and non-toxic solution for assessing bacterial efflux for your research on antibiotic resistance. This coloured fluorescent compound quickly accumulates within bacteria as a function of their efflux pump activity. Simply add ColorFlux to your bacteria and watch them change colour !

How does ColorFlux work?

ColorFlux staining was shown to reflect the activity of well-characterized efflux pumps from the major facilitator superfamily and ATP-binding cassette families in a variety of Gram+ and Gram- bacteria:
NorA, MepA, MepB, PatA, PatB (Staphylococcus aureus & Streptococcus pneumoniae)
BmrA (Bacillus subtilis)
AcrAB (Haemophilus influenzae)
The list will be updated regularly according to the feedback of users.

Technical information about ColorFlux

Tested and validated on : Staphylococcus aureus, Bacillus subtilis, Streptococcus pneumoniae, Haemophilus influenzae.
Colour: Red
Form: liquid
Detection methods: visual & fluorescence. Also compatible with single-cell approaches (live imaging and flow cytometry)
Peak excitation/emission wavelengths: Ex:530nm/Em:650nm
Storage: at 4°C protected from light for 8 months
1 kit contains 1mL of an aqueous solution with a concentration of 1 mg/mL for a final volume of 1L of screening medium

ColorFlux staining of MepA&B mutants in Staphylococcus aureus

Left graph shows the fluorescence signals detected for each strain using an Ex=530nm, and right graph shows corresponding fluorescence intensity values obtained for each strain at Em=645nm. WT = Wild Type strains, MepA1B+++ = MepA&B overexpressing strains.
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of NorA mutants in Staphylococcus aureus

Top picture shows pellet colour variations according to NorA expression. Bottom figures show the fluorescence signals detected for each strain using an Ex=530nm.
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of PatA/PatB mutants in Streptococcus pneumoniae

Left pictures show pellet colour variations according to PatA/PatB expression. The right graph show fluorescence signals detected for each strain using an Ex=530nm.
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

ColorFlux staining of BmrA mutants in Bacillus Subtilis

Top picture shows pellet colour variations according to BmrA expression. Bottom figures show the fluorescence signals detected for each strain using an Ex=530nm.
Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

Distinguishing efflux activity levels across H. influenzae strains

Various strains of H. influenzae with known efflux activity were incubated with 1 µg/mL ColorFlux for 25 minutes and centrifuged.

Credits: Cameron Huhn – University of Mississippi Medical School, 2024

Bacillus subtilis bacteria (moderate activity) stained with ColorFlux

Credits: Mrunal Patil & Jean-Michel Bolla, Aix-Marseille Université – 2023

How to use ColorFlux bacterial efflux probe:

Find below advice & tips for ColorFlux use:

How does ColorFlux work?

ColorFlux is a cationic compound that quickly penetrates Gram + bacteria through passive diffusion and is efficiently expelled out of the cell depending on efflux activity.

How is efflux level measured?

After incubating bacteria with ColorFlux for 15 minutes, a steady-state level is reached and relative levels of efflux activity can be quickly assessed by looking at cell pellet color. It is also possible to read fluorescent signals for a precise quantification of efflux levels. Alternatively, kinetics of ColorFlux accumulation can be monitored by flow cytometry.

How reliable is ColorFlux for detecting efflux-mediated resistance?

ColorFlux is a substrate for efflux pumps that are well-known as key players in bacterial efflux-mediated resistance. In particular, NorA involved in first-line response in S. aureus, PatA/B involved in fluoroquinolone resistance in S. pneumoniae or BmrA, a well-characterized multidrug ABC transporter in B. subtilis.

In a pilot study conducted at IHMT-NOVA, Lisboa on 101 S. aureus clinical strains from humans and animals, ColorFlux detected efflux-resistant bacteria simply by colorimetric assessment. This study demonstrated that ColorFlux can be used as a quick & reliable tool to detect clinically relevant resistant bacteria.

Which efflux pumps is ColorFlux a substrate of?

ColorFlux staining was shown to reflect the activity of well-characterized efflux pumps from the major facilitator superfamily and ATP-binding cassette families in a variety of Gram+ bacteria:

NorA, MepA, MepB, PatA, PatB (Staphylococcus aureus & Streptococcus pneumoniae)
BmrA (Bacillus Subtilis)
We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Is ColorFlux safe to handle?

Contrary to ethidium bromide, ColorFlux is a non-toxic compound that you can safely handle on the bench and dispose of. It is a particularly useful tool for training purposes & on-field applications.

How long will it take to measure efflux levels?

The total procedure to stain bacteria and visualise efflux levels by colorimetric assessment should take no more than 30 minutes.

Can I use ColorFlux in Gram- bacteria?

ColorFlux is primarily recommended for Gram+ bacteria as ColorFlux proved efficient at reflecting relative levels of efflux pump activity in all species tested so far. Indeed, the fast influx/slow efflux properties of Gram+ bacteria allow ColorFlux to be quickly internalized and then expelled as a function of efflux, resulting in different shades of red that are representative of relative levels of efflux activity.

Because Gram- bacteria tend to have a slow influx/fast efflux profile, the concentration of ColorFlux and incubation time might need to be adjusted for each particular bacterial species and efflux pump involved. So far, ColorFlux has been successfully used to visually distinguish relative efflux levels of the AcrAB pump in mutant Haemophilus influenzae bacterial strains using the recommended protocol of use. For other species, such as E.coli, some preliminary tests combining ColorFlux with compounds to reduce efflux pump activity, i.e. CCCP, have shown promising results and might help getting relative efflux level measurements.

To sum up, the applicability of ColorFlux in Gram- bacteria will depend on the intended application. While ColorFlux can be useful as a tool to quickly check for the presence/absence of efflux pumps (i.e. to validate efflux pump knock-out mutants), some protocol optimization will be required if you are aiming at comparing relative efflux activity levels between strains, or to detect resistant bacteria.

DNAbsolute - A column-free DNA extraction buffer

DNAbsolute is a precipitating reagent for quick & safe DNA extraction of a wide range of samples

DNAbsolute provides a fast & safe method to isolate DNA from a wide range of sample types. Its high sensitivity makes it a solution of choice when working with small & precious samples.

Products

Product Product Size Catalog No.
DNAbsolute – column-free DNA extraction buffer 5ml ABS-5-IDY
DNAbsolute – column-free DNA extraction buffer 25ml  ABS-25-IDY
DNAbsolute – column-free DNA extraction buffer 50ml ABS-50-IDY

Safe & simple
Based on ionic liquids, the DNAbsolute method does not involve any plastic columns nor harmful chemicals. DNAbsolute is the go-to solution for researchers seeking a rapid, user-friendly, and eco-conscious DNA extraction solution.

​Versatile
Since its release, DNAbsolute has been successfully used to extract DNA from a wide panel of samples, including some of the most challenging ones:

Insects (drosophila, psyllids, ants, beetles, butterflies, cochineals)
Fish (shark fins)
Mammals (rodent skin)
Plants
Corals
Bacteria (Gram -)
Mollusks
Cnidarians
Protists

Sensitive
Allowing efficient DNA recovery from single specimens down to a single leg, DNAbsolute is a solution of choice to get the most of museum specimens, limited or precious samples.

Kit description of DNAbsolute

5mL kit

25 to 250 extractions*

5mL DNAbsolute 3M

5mL PBS 150 mM, pH 7.2

5mL Tris 25 mM, pH 8

12mL Lysis buffer 1

12mL Lysis buffer 2

25mL kit

125 to 1,250 extractions*

25mL DNAbsolute 3M

25mL PBS 150 mM, pH 7.2

25mL Tris 25 mM, pH 8

2x 25mL Lysis buffer 1

2x 25mL Lysis buffer 2

50mL kit

250 to 2,500 extractions*

50mL DNAbsolute 3M

2x 25mL PBS 150 mM, pH 7.2

2x 25mL Tris 25 mM, pH 8

2x 50mL Lysis buffer 1

2x 50mL Lysis buffer 2

*The number of extractions per kit depends on your starting sample weight. For samples weighting from 50 mg to 100 mg, refer to the lowest number of extractions provided. This number will then be inversely proportional to the weight, and can be multiplied by up to 10 when working with samples under 10 mg.

Storage: PBS, Tris & Lysis buffers are stable for 1 year at room temperature (15-25°C). The 3M DNAbsolute reagent shoud be stored at 4°C and is stable for one year.

DNAbsolute is a visual method: check out your DNA at the first step of the protocol

DNA pellet obtained from 50 drosophila after DNAbsolute precipitation

 

Extraction of PCR-grade DNA using DNAbsolute

DNA was extracted from 50 drosophila using either a column-based commercial kit (left column) or DNAbsolute (middle and right columns). The resulting DNA was used for PCR amplification of mTOR gene and amplified products were run on an electrophoresis gel.

 

Extraction of highly pure genomic DNA from drosophila using DNAbsolute

DNA was extracted from 50 drosophila using either a column-based commercial kit (left column) or DNAbsolute (right column). The resulting DNA was run on an electrophoresis gel.

Extraction of highly pure DNA from coral specimens using DNAbsolute

Credits: Gabriel Johnson – Smithsonian Institution, Museum Support Center, Maryland, US

 

Extraction of high DNA yields from dried plant leaves using DNAbsolute

Plant leaves either from herbarium specimens (canellaceae – canellaceae & pleodendron) or dried using silica gel (Nyctaginaceae – Pisonia) were extracted using DNAbsolute and run on an agarose gel. DNA yields were quantified on a Qubit instrument.

Credits: Gabriel Johnson – Smithsonian Institution, Museum Support Center, Maryland, US

 

PCR amplification of genomic DNA extracted from red coral specimens

DNA from ethanol-preserved (1-6) or dried (7-8) Corallium rubrum specimens was extracted using DNAbsolute and diluted 1:10 before serving for PCR amplification of a genomic portion.

Credits: Eugénie Kreckelbergh – Université d’Aix-Marseille, MIO (Institut Méditerranéen d’Océanographie) et FAO (Food and Agriculture Organization of the United Nations).

How to use DNAbsolute:

How does DNAbsolute work?

DNAbsolute is composed of a ionic liquid that has a selective and high affinity for DNA strands. When mixed with your sample, the DNAbsolute reagent will directly bind DNA and precipitate it without the need for RNase treatment, protein precipitation or use of hazardous reagents.

Which types of samples can DNAbsolute extract DNA from?

DNAbsolute has been successfully used for extracting DNA from:

  • Insects: drosophila, psyllids, beetles, butterflies, ants, cochineals
  • Plants: tomato, canellaceae, santalaceae, loranthaceae, nyctaginaceae, inula, pistacia, phyllirea
  • Corals: corallium rubrum
  • Bacteria: Salmonella & e.coli
  • Fish: shark fins
  • Mammals: rodent skin
  • Mollusks: mussels, whelks
  • Cnidarians: Bunodeopsis strumosa
  • Protists: stramenopiles

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Can I use DNAbsolute to extract DNA from dry samples?

Yes, DNAbsolute is efficient to isolate DNA from dried samples. So far, DNAbsolute has shown efficient DNA extraction from dried corals and plants (either herbarium specimens, or fresh material dried using a freeze-dry method or silica gel).

Can I use DNAbsolute to recover high molecular weight DNA?

Yes, DNAbsolute is efficient at recovering HMW DNA fragments ( >48kb for dried corals, >24kb for arthropods).

How pure will the extracted DNA be?

DNAbsolute DNA extraction method can yield highly pure DNA due to its ability to efficiently and specifically solubilize DNA, minimizing contamination with proteins, RNA, and other cellular debris. The purity of drosophila and coral DNA extracted using DNAbsolute has been assessed by spectroscopic measurements. The average 260/280 ratio was found between 1.8-1.9 and the 260/230 ratio at 2.3.

The type of biological sample being processed can affect the extracted DNA purity. Sample lysis optimization, or additional purification steps, might be implemented when working with samples containing high levels of secondary metabolites.

How can I check that the DNA extraction was successful?

A white pellet visible to the naked eye should form immediately after adding the DNAbsolute reagent to your sample lysate. This way, you can easily and rapidly check if your DNA successfully precipitated before proceeding to purification and washing steps.

Are there any requirements on how concentrated/pure my starting material needs to be?

As a general rule, DNAbsolute is efficient at extracting DNA from a starting solution with a concentration of at least 5 ng/mL. DNAbsolute proved successful at extracting DNA from small starting material, including individual insect specimens (cochineals, psyllids & ants), a couple of legs (butterfly) and even down to a single leg (beetles). As an example, DNA yields from fresh individual cochineals were between 5-155 ng/µL, and between 5-400 ng/µL for a group of 6 individuals.

Moreover, the high specificity of DNAbsolute reagent for DNA means you can safely get pure DNA without additional steps of RNase treatment or protein precipitation, so DNAbsolute is an interesting option if your starting material is not the cleanest.

DNAbsolute is efficient at recovering DNA fragments >300 bp, and will therefore work best with fresh material or material that has been stored immediately at -20°C or below with limited thawing & freezing cycles.

How long will it take to extract DNA using DNAbsolute?

Once your sample is lysed, the whole DNA extraction process should take no more than 30 minutes.

What is included in the DNAbsolute kit? Is there anything I need that is not provided?

Our DNAbsolute kit contains the DNAbsolute solution as well as some lysis, PBS and Tris buffers. All you need to have on your side is a refrigerated centrifuge, ethanol and proteinase K. You can also add an RNase treatment if needed.

Can I use another lysis buffer than the one supplied in the DNAbsolute kit?

The DNAbsolute reagent is used to purify DNA from a whole lysate. The lysis buffer provided in the DNAbsolute kit is suitable for lysing a wide range of sample types (Gram – bacteria, soil, whole organisms, etc) prior to DNA purification with DNAbsolute. Alternatively, there is no problem in using your own buffer and method of choice for lysis. You can then add the DNAbsolute reagent to your lysate to purify the DNA.

How many experiments can I carry out with one DNAbsolute kit?

DNAbsolute is available as a 5 mL, 25 mL or 50 mL solution allowing respectively up to 250, 1,250 or 2,500 extractions. Those numbers have been calculated based on 50 drosophila per extraction, and need to be adjusted depending on the quantity and volumes of your starting material while respecting the proportionality of the buffers used.

Can DNAbsolute be used for plant DNA extraction?

Yes, DNAbsolute has been successfully used to extract plant DNA from dry leaves (either herbarium specimens, leaves dried using a silica gel or freeze-dried). The method works very well with low starting material (down to 1.5 mg, or a single disk leaf), and the obtained DNA has been successfully used for PCR applications. It is useful to keep in mind that the obtained DNA quality will be very much dependent on your starting material quality, preparation & lysis methods. If particularly high quality standards are needed for your downstream application (i.e. sequencing) it could be that some protocol adjustments are needed (for example, additional subsequent purification steps).

What are DNAbsolute applications in marine biology?

Since its initial launch, DNAbsolute has been heavily used for marine environmental biology. Indeed, DNA extracted from coral specimens (both ethanol-preserved or dried), mussels or even cnidarians. The extracted DNA was directly used for both PCR and qPCR assays after a 1:10 dilution.

What yield can I expect?

The resulting yield can vary widely depending on the sample type and preparation. As a general rule, you can expect to recover on average 85% of DNA from a solution ranging from 10 to 100 μg/mL of DNA, and 60% of a solution ranging from 5 to 10 ng/mL of DNA. Poor quality, and/or fragmented starting material will result in reduced yield of purified DNA.

Here are some yields obtained for different types of samples:

Can DNAbsolute be used for RNA isolation?

The DNAbsolute ionic liquid has been specifically optimized for double-stranded DNA recovery. We do not recommend using it for RNA isolation.

UbiClear - clearing kit for biological tissues

Kit contents:
– 1x Depigmentation Mix
– 1x Clearing Base
– 1x Imaging Solution

UbiClear is an innovative method for optical clearing of biological samples. Thanks to its unique formulation, the UbiClear solution makes thick samples transparent in less than 20 minutes without any toxic compounds. Simply place your sample in the clearing buffer, incubate at 37°C and transfer it to the imaging solution. A great way to make clearing accessible to anyone!

Ready to dive into deep tissue imaging ?

Non-toxic
Formulated without any harsh chemicals, UbiClear can be handled safely and does not deteriorate microscope objectives

Compatible with fluorescence labeling
UbiClear preserves endogenous fluorescence and can be combined with immunostaining protocols

 Ultra-fast
Stop spending days on clearing: clear your samples within minutes thanks to a 1-step clearing method

Versatile
Clears a vast panel of soft & hard mammalian tissues (bone, teeth), insects & plants. Nothing can stop it!

How does UbiClear work?

UbiClear is an aqueous solution that modifies the optical properties of complex biological tissues by removing lipids and homogenizing refractive indexes. This renders opaque tissues transparent while keeping their original structure intact. As a result, light passes through specimens without any scattering or absorption, yielding high-quality images of entire 3D structures. The UbiClear workflow is straight-forward, and does not require any specific expertise or access to sophisticated equipment.

Step 1
Remove sample coloration by immersing it into UbiClear Depigmentation Buffer for 1h at 37°C.

Step 2
Clearing (20min – overnight)
Incubate your sample into the UbiClear clearing solution at 37°C until fully transparent.

Maximal transparency is achieved in a few minutes for most tissue fragments (<20 min for mouse cerebral cortex and small intestine), and may take up to a few hours for entire organisms.

Step 3
Imaging
Transfer your cleared sample to the imaging solution (RI = 1.47) for imaging and maintaining transparency on the long-term.

Step 4
Repeat !
If needed, samples can be re-opacified simply by transferring them to 0.2 M Tris buffer.

Discover more on Ubiclear & its applications by hearing directly from its inventors: http://brg.sppin.fr

 

UbiClear applications

Sample types: UbiClear can be used to clear a wide spectrum of samples ranging from small 3D cell structures up to entire organisms. So far, UbiClear has shown efficient clearing of:

– 3D cell structures: human embryoid bodies, human mini-brains

– Human tissues: frontal cortex

– Mice tissue sections (up to 1 mm thick): spinal cord, brain (cerebellum, choroid plexus), testes, seminal vesicle, ear (elastic cartilage and hair), lungs, heart, intestine, kidney, skeletal muscle, yolk sac membranes

– Ox tissues (up to 1 mm thick): knee (joint cartilage and bone)*

– Plants: ivy leaves, carrot slices, beet roots, microcallus & callus organoids, nasturtium stamen, melon seeds, sprouted tomato seeds, rose stem slices

– Whole organs: mouse brain, heart, intestine, eye, teeth*, dorsal root ganglia

– Whole organisms: Colisa Lalia, adult zebrafish, firebugs (Pyrrhocoris apterus), wasps, grasshoppers, shield bugs (Graphosoma lineatum), flies, cockroaches, spiders

– Crustaceans: woodlouse

– Molluscs: mussel (including shell)*

*for these samples, an additional step of demineralization needs to be performed before clearing.

 

Compatible assays: label-free anatomical studies (autofluorescence, volumetric imaging), dye-based assays (Evans blue, albumin-FITC), immunostaining. UbiClear preserves endogenous fluorescence.

Microscopy techniques: confocal, spinning-disk, light-sheet microscopes*.

*Extra diluting buffer can be purchased separately to make more imaging solution if needed (i.e. for use with light sheet microscopes).

 How to use UbiClear:

Storage: The depigmentation buffer, Mix 1 and Mix 2 solutions should be kept at room temperature, protected from light. In these conditions, all solutions can be stored for at least 1 year.

Kit contents of UbiClear:

 

Small kit
Depigmentation Buffer: 20 mL Mix 1: 5 mL Mix 2: 40 mL
Final volume of depigmentation, clearing & imaging solutions = 20 mL each*

 

Large kit
Depigmentation Buffer: 200 mL Mix 1: 50 mL Mix 2: 400 mL
Final volume of depigmentation, clearing & imaging solutions = 200 mL each*

*Mix 1 & Mix 2 are mixed at a 1:5 ratio to make the final clearing solution. Mix 2 will be added with water to make up the final imaging solution.

An extra 400 mL volume of Mix 2 can be purchased separately to make more imaging solution if needed (i.e. to fill out light sheet microscope tanks). A final volume of 400-500 mL of imaging solution can be prepared using the 400 mL of Mix 2 provided.

The volume of clearing solution required varies according to your sample size. As a general rule, we recommend using a clearing solution volume corresponding to 10X your sample volume. Please refer to the table below to find out which kit format will best suit your needs.

Fast & versatile clearing of diverse animal tissues using UbiClear

1 mm thick mice brain slices (fat tissue), 2 cm mice intestine fragments and a 1 mm thick ox knee slices (joint cartilage and bone, calcified tissue) were cleared using the UbiClear method and imaged on a striped target. Ox knee joint samples were decalcified and all samples depigmented and cleared using UbiClear. The target stripes are visible through the sample after clearing for less than an hour.
Credits: Brigitte Delhomme & Martin Oheim (CNRS, SPPIN UMR8003)

Label-free 3D morphological characterization of the deep enteric nervous system

A 1.5 cm long mice small intestine fragment was cleared using UbiClear and deep autofluorescence images were taken using a two-photon microscope at different depths of the intestine wall, starting from the muscularis longitudinalis down to the crypts. The video shows the 3D rendering and animation of the fine architecture of the enteric nervous system based on a z-stack acquired (800 nm excitation, z-step: 1 µm, 480 ms/image).
Source publication: Hazart D. et al, 2023

Visualizing Glial fibrillary acidic protein (GFAP) expression in mouse brain

Indirect immunofluorescence and clearing was performed on 1 mm thick sections of formaldehyde-fixed mouse brain using GFAP antibody and secondary antibody Alexa 555 fluorophore. On the left the whole slice was imaged in mosaic with 10XNa0.3 objective, on the right 40XNa1.0 was imaged in the cortex of the mouse brain.
Credits: Diane Derrien, Brigitte Delhomme & Martin Oheim (CNRS, SPPIN UMR8003)

Cleared mouse dorsal root ganglia combined with immunohistochemistry

Immunofluorescence and clearing was performed on whole fixed dorsal root ganglia from a transgenic mouse model that expresses both (i) a genetically-encoded GCaMP6 calcium sensor in sensory neurons as well as (ii) a hematoglutinin-tagged chemogenetic receptor in satellite glial cells. An anti- GFP (green) and an anti-hematoglutinin (red) antibodies were used to amplify GCaMP6 staining and reveal satellite glial cells, respectively. The whole ganglia was imaged using a confocal microscope Axio LSM710 (Zeiss) and 10XNa0.3 WD 5mm air objective, in 620 µm depth and z 2 µm. 3D reconstruction was obtained with Imaris viewver software (oxford instruments).

Credits: Andreia Ramos (Université Paris Cité, CNRS, INCC UMR8002, Cendra Agulhon’s Group) and , Brigitte .Delhomme (Université Paris Cité, CNRS, SPPIN UMR8003, Martin Oheim’s laboratory). Courtesy of Cendra Agulhon.

Mapping Iba1 expression in a cleared mouse spinal cord

Immunofluorescence was performed on a 500µm mouse spinal cord section after depigmentation. Iba1 antibody, a rabbit polyclonal antibody, and Cy3 secondary antibody were used for incubations. Iba1 was imaged using an Axio LSM710 (Zeiss) confocal microscope with 10XNa0.3 WD 5mm air objective, zoom 0.6 in 270µm depth and z 2µm. 3D reconstruction was performed using Imaris viewver software (Oxford Instruments).
Credit : Clearing B.Delhomme & M.Oheim (CNRS, SPPIN UMR8003)

Insect clearing using UbiClear

A firebug (Pyrrhocoris apterus) was depigmented, embedded in low melting agarose and cleared using UbiClear. The image showing autofluorescence signal at 488 nm was acquired using a light-sheet ultramicroscope (Labvision). 2,400 images were stitched for the 3D reconstruction.
Credits: Clearing: Brigitte Delhomme (CNRS, SPPIN UMR 8003), Imaging: J. Fernandes (Institut Pasteur, Paris) & Martin Oheim (CNRS, SPPIN UMR8003)

Whole brain clearing using UbiClear

A whole brain from a 4-month old mouse was depigmented and cleared using UbiClear. The picture was taken 48h after clearing.
Credits: Brigitte Delhomme & Marwa Moulzir (CNRS, SPPIN UMR 8003)

One-step clearing of the small intestine

A 1.5 cm long mice intestinal fragment was cleared using UbiClear and placed in a home-made tank with a black-and-white stripe pattern. Images were acquired on a SMZ800 stereo macroscope (Nikon, Amstelveen, The Netherlands) after 10sec, 110sec and 20min of incubation in the UbiClear clearing solution. Michelson’s contrast calculation revealed that 71% transparency was reached in less than 15 min.
Source publication: Hazart D. et al, 2023

Imaging deep blood vessel network in mouse cerebellum

Mouse was perfused with FITC albumin and sacrificed to image blood vessels in the cerebellum. A 500µm tissue slice was cleared without depigmentation and imaged using an Axio LSM710 (Zeiss) confocal microscope with 488nm laser and 10XNa0.3 WD 5mm air objective, in 508µm depth and z 4µm. 3D reconstruction was performed using Imaris viewer software (Oxford Instruments).
Credits : Mouse painting vessels X.Bai (University of Saarland, Homburg (DE)), Clearing B.Delhomme (CNRS, SPPIN UMR8003, Paris (Fr)) , F.Licata (UMS Paris-cité University (Fr) & Martin Oheim (CNRS, SPPIN UMR8003, Paris (Fr))

Detailed morphological characterization of the enteric nervous system

Mice small intestine fragments were cleared using UbiClear and immunolabeled against a pan-neuronal marker (red) and an enteric-glia marker (green). Single autofluorescent images (grayscale) and fluorescence images were taken at different depths in the intestinal wall (muscularis longitudinalis, Auerbach’s plexus, Meissner’s plexus and crypts). Images were acquired on a LSM880 inverted confocal Airyscan microscope (Zeiss).
Source publication: Hazart D. et al, 2023

Publications using UbiClear:

Hazart D, Delhomme B, Oheim M and Ricard C (2023) Label-free, fast, 2-photon volume imaging of the organization of neurons and glia in the enteric nervous system. Front. Neuroanat. 16:1070062. doi: 10.3389/fnana.2022.1070062.

Doriane Hazart, Malvyne Rolli-Derkinderen, Brigitte Delhomme, Pascal Derkinderen, Martin Oheim and Clément Ricard (2024) L’intestin, lanceur d’alerte, dans les prémices de la maladie de Parkinson. Med Sci (Paris), 40 6-7 (2024) 544-549. doi: https://doi.org/10.1051/medsci/2024082

 

Discover all posters featuring UbiClear at the AFH (French Association of Histotechnology) congress:

How is UbiClear better than other clearing methods?

UbiClear relies on an aqueous clearing solution, formulated without any harmful solvents or expensive components, allowing to make thick biological samples transparent in no time. By providing a safe and affordable technique even when large volumes are needed, UbiClear was designed to democratize tissue clearing for researchers interested in deep tissue imaging.

Which types of samples is UbiClear efficient for?

UbiClear can be used to clear a wide spectrum of samples ranging from small 3D cell structures up to entire organisms. So far, UbiClear has shown efficient clearing of:
– 3D cell structures: human embryoid bodies, human mini-brains.
– Human tissues: frontal cortex.
– Mice tissue sections (up to 1mm thick): spinal cord, brain (cerebellum, choroid plexus), testes, seminal vesicle, ear (elastic cartilage and hair), lungs, heart, intestine, kidney, skeletal muscle, yolk sac membranes.
– Ox tissues: knee (joint cartilage and bone)*.
– Fish tissues: Colisa Lalia, zebrafish.
– Plants: ivy leaves, carrot slices, beet roots, microcallus & callus organoids, nasturtium stamen, melon seeds.
– Whole organs: mouse brain, heart, intestine, eye, teeth* and dorsal root ganglia.
– Insects: firebugs (Pyrrhocoris apterus), wasps, grasshoppers, spiders, shield bugs (Graphosoma lineatum), flies, cockroaches.
– Crustaceans: woodlouse.

*for bone & teeth samples, an additional step of demineralization needs to be performed prior clearing.

We would greatly appreciate your valuable inputs by helping us complementing this list with any of your positive research experiences. Drop us a message through our Contact page!

Is clearing reversible?

Yes, it is possible to re-opacify the samples by incubating them in a 0.2 M Tris buffer at pH 8. Re-opacifying can be a practical way to select and section a region of interest for downstream assays (i.e. immunostaining).

What is the refractive index of the Imaging Solution?

The final imaging solution obtained following our protocol of use (i.e. diluting the Mix 2 solution in water) has a refractive index of 1.47. It is possible to increase it up to 1.485 by adding less water to the Mix 2 solution.

How does UbiClear work?

UbiClear efficiently clears biological tissues by removing lipids (Mix 1) and homogenizing refractive indexes (Mix 2) of the sample’s internal components. The clearing solution is prepared by mixing Mix 1 & Mix 2, and the imaging solution is prepared by diluting Mix 2 in water.

Will I be able to visualize endogenous fluorescence after clearing with UbiClear?

Yes, endogenous fluorescence is preserved upon clearing (this has been shown with dT-tomato and GFP). If your sample contains a fluorescent native protein, you simply need to skip the optional depigmentation step as this will likely interfere with the fluorescence signal.

Is it possible to combine clearing with immunostaining protocols?

Yes, it is possible to perform immunostaining and clear your labeled sample with UbiClear afterwards.
Immunostaining using AF405, AF488, AF555, AF647, Cy3, TMR, AM-cyan &Td tomato have been successfully performed on samples cleared with UbiClear.

Can I combine clearing with direct dye labeling?

Yes, UbiClear can be combined with direct dyes for visualizing cellular or structural features. This has been validated with Evans blue and albumin-FITC for imaging blood vessels in cleared samples. UbiClear is not compatible with common direct nucleic acid dyes (DAPI, BET, methyl green, Topro3…). If DNA staining is expected, we recommend using indirect antibody labeling strategies.

Which fixation methods is UbiClear compatible with?

So far, the UbiClear kit has been successfully used on samples cleared using 10% formalin (or 4% PFA), and that is therefore the fixation method we recommend. It could be that clearing works well with other fixation solutions, yet we can’t guarantee that clearing efficiency will be similar.

How should I image my cleared samples?

Once cleared, samples can be imaged using conventional confocal microscopes (for samples up to ~500 µm thick). Thicker samples may be imaged using light-sheet microscopes.
Need some advice? We would be happy to answer any questions on the choice of microscopy technique, data analysis and handling. Feel free to reach out at technical@stratech.co.uk

How long will it take to clear my sample with UbiClear?

Total duration will vary depending on your sample thickness and internal properties. As a general rule, the total procedure should not take more than 2 hours for samples up to 1 mm thick. The protocol might be extended up to a few hours for entire organisms.

What is UbiClear not suitable for?

As UbiClear works by removing lipids from the samples, this solution will not be suitable for imaging & staining lipids. It is also not compatible with direct nucleic acid dyes (DAPI, BET, methyl green & Topro3). If DNA staining is expected, we recommend using indirect antibody labeling strategies.

Will my sample shrink upon clearing?

Sample shrinkage has been observed within the first few minutes of incubation in the clearing solution, which is explained by the difference in osmolarity between the sample and the solution leading to dehydration. This effect is then attenuated after a few minutes as the clearing solution penetrates inside the structures.

Is UbiClear toxic?

No, UbiClear does not contain any organic solvents neither volatile compounds. Its handling does not require the use of a chemical hood or sophisticated ventilation systems for imaging.

What is included in the UbiClear kit, and what should be provided by the user?

The UbiClear kits include a Depigmentation Mix, a Clearing Base and some Imaging Solution. Everything you need to have on your end is some water, 0.2M Tris at pH 8 and some 30% hydrogen peroxyde.

How many experiments can I perform with one kit?

The volume of clearing solution required varies according to your sample size. As a general rule, we recommend using a clearing solution volume corresponding to 10X your sample volume. Please refer to the table below to find out which kit format will best suit your needs.