Simplified protocol & conditions of success

5 key conditions of success:

Method of use:

Actiflash and us

” Once upon a time, a physicist (David Bensimon) asked a chemist (Ludovic Jullien) whether he could design a caged inducer to photocontrol protein activity in living organisms. For sure! However we also needed a biologist (Sophie Vriz) to accept the challenge to validate the caged Cyclofen-OH technology. It has been a long but so nice adventure, which has involved the tight integration of the work from many talented students, postdocs, and collaborators… Thanks to all of them!”

Esther Graudens, New projects, Idylle:

“Actiflash perfectly highlights the researchers’ need to calibrate the experimental conditions in life sciences. We wanted to initiate a global scheme on this topic and find solutions to help them get reproducible results. So, when we met Ludovic Jullien’s team, it was obvious to us that Actiflash was a first step in the right direction. Their technology had already been tested and approved by a large number of researchers. It was then a question of making it a stable product, of producing it and disseminating it to the research communities. It is a great satisfaction to propose it today and we are very proud that Ludovic Jullien’s team has chosen to widespread its technology with Idylle! Ludovic has extensive experience in transfer options, and his choice also reinforces our approach of promoting the know-how of researchers!”

Chitozen: how to use it, a video protocol from assembling the elements to imaging your favorite bacteria.

The material you will need:
Hardware

µ-Slide Microscopy Rack
Eppendorf® Centrifuge 5430 & Rotor 5430R A-2-MTP
Clamp and adapter for sticky slides

Consumables

Bottomless 6 channels sticky slides (sterilized and welded in a gas-permeable packaging)

Reagents

LB medium
LB ½ medium
Milli-Q® water

Cells

1. coli – Mycobacterium smegmatis – Bacillus subtilis – Pseudomonas fluorescens – Vibrio cholerae –  Myxococcus xanthus – Pseudomonas aeruginosa.

Chitozen coverslips allow bacterial cells to be immobilized even under a flow.

Storage conditions: 

• Everspark has to be stored in the fridge at 4°C upon receiving the product. Do not freeze it.
• Open the tube just before mounting to limit oxygenation
• Store the slides at 4°C and in the dark before repeating dSTORM acquisition on the same slide.

Use method:

• Rinse the coverslip with sterile water
• Discard water in excess
• Prepare sealant: 1 volume of sealant catalyst + 1 volume of sealant basis in a micro-dish – do not mix
• Open the Everspark tube and pipette right away the desired volume onto a Wilco glass bottom dish
• Flip the coverslips face towards the slide on the cavity filled with Everspark without making bubbles
• Absorb excess of Everspark buffer on the sides with a kimwipe
• Mix sealant
• Seal quickly with the reconstituted sealant
• Wait for the sealant to polymerize on the slide before moving the slide
• Discard the open tube of Everspark buffer (cannot be used for another slide, once already opened)
• Gently clean the coverslip with 70% ethanol
• Perform regular dSTORM acquisition (best to wait a couple of hours)
• Repeat dSTORM acquisition 24 hours up to 3 weeks on the same slide providing no air bubbles were present
• Store the slides at 4°C and in the dark before repeating dSTORM acquisition on the same slide (up to 3 weeks)

Note 1: Blinking events have been validated with JF646, JF549, AF647, CF647, DL550, CF568, DL650, CF680, SulfoCy5 and mEos2

Note 2: Blinking is detected for several weeks without loss of events or photons per event

Note 3: Mounting can also be performed in various microdishes by adjusting the Everspark volume (Each vial contains 450 µl)

See the technical information about each shape in details.

I successfully managed to use Stampwell with Collagen-1.
Matthieu Opitz • Alveole, France

I use the Stampwell V-shape to track the explants development and the embryoids formation from stem cell aggregates in zebrafish, and it makes my work much easier!
Queralt Tolosa • Centre for Genomic Regulation, Spain

I used a Stampwell to ensure that my zebrafish embryos were positioned correctly under the microscope for spatial and temporal experiments. I am pleased with my Stampwell because it saves me time and provides precision!
Shai Eyal • UCSD, USA

Stampwell has proven to be a very easy to use mounting medium for confocal imaging of fixed and immunostained zebrafish embryos.​
Matthieu Simion • TEFOR, France

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, Spain

Utilizing the Stampwell V-shape when turning airway organoids inside out is useful to keep them separated, therefore avoiding clumping and merging.
Signe Lolle • Danmarks Tekniske Universitet, Denmark

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

Imprinted V-shape 300µ well in agarose
Soaked in fluorescein, negative rendering
Image credit: (c)Gaëlle Recher – Bordeaux

Cell-filled alginate capsule in the V-shape well, imaged in z
Notice that the focus moving through the well height is easily visible (lines in focus Vs out of focus)
Image credit: (c)Gaëlle Recher – Bordeaux

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

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

1

Pour liquid agarose

2

Place the stamp

3

Reticulate the hydrogel

4

Remove the stamp

To image, just load your samples and image, sequence, transport at will.

To aggregate, seed your cells & let them aggregate.

V-shape & Embryo 1 specific protocol

Resources

• The most Frequently Asked Questions (FAQ)
• The V-shapes and Embryo 1 Datasheet
• The Embryo 2 Datasheet
• The Larvae 1 Datasheet
• The Larvae 2 Datasheet

Kit description

• Diameter of the stamp: 26mm
• 6 shapes of wells to choose freely to fit your experiments. See below.

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

Embryo 1 (aka 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: 10. 5 pins for zebrafish larvae and 5 pins for the medaka larvae on the same stamp
. Shape of the pins: a prism.
. Length of the wells: 10 mm for the 5 long wells and 3 mm for the 5 small wells.
. Depth: 1 mm
. Width at the bottom of the wells: 0.3 mm
. Width of the upper side of the wells: 2 mm

Storage conditions 

• The Stencells are packaged between 2 protective foils: if you wish to store them, keep them between these foils, in order to avoid dust deposition.
• Storage at room-temperature, stable up to 2 years.

Key conditions of success

• When you remove the inner elements of your Stencell, use 2 tweezers: 1 to remove and the other 1 to press close to the junction in order to avoid breaking the Stencell. Alternatively, you can cut the junctions with a scalpel.
• When you put the Stencell on top of your culture substrate, softly remove the bubbles and stick the Stencell, patting it with tweezers
• Depending on the substrate hydrophobicity, the droplet might not completely fill the well. You can:
  * Either fill the well with the standard volume. Then, help the droplets stick to the Stencell by connecting the liquid next to the Stencell walls using the pipette tip.
  * Or, you may add an excess volume of liquid (e.g. 30 μL) and remove it afterwards (e.g. 10 μL).
• But: do not overfill (e.g. > 30 μL) the wells, otherwise two droplets may merge.
• When you put your substrate and cells in the incubator. Wait for 1 to 2 hours until cells spread and fully occupy the windows space. If you need to wait longer, be sure  that the droplets do not completely evaporate

Protocol of use

• Wound-healing experiment in Stencell chambers
  1)     Using tweezers, discard the windows of a Stencell
  2)     Using tweezers, grab the Stencell and place it on your dedicated cell culture substrate (glass coverslip, plastic Petri dish, multiwell plate)
  3)     Optional: use a flat surface to make sure the Stencell properly stick to the cell culture substrate.
  4)     Immerse the sample in complete cell culture medium and place it in an incubator (37 °C, 5 % CO2).
  5)     Detach your cells according to your standard procedure, centrifuge.
  6)     Remove the bubbles present at the interface Stencell/cell cuture substrate using a pipette.
  7)     Seed the cells and place the sample back in the incubator until cells are spread and fully occupy the windows space.
  8)     Carefully remove the Stencell using tweezers while maintaining one corner with a pipette.
  9)     Optional: Place the glass coverslip on its dedicated holder.
  10)   Image with a microscope at 37 °C, 5 % CO2.
• lmmunostaining of cells confined in Stencell chambers
  1)  Using tweezers, discard the windows of your Stencell
  2)  Using tweezers, grab the Stencell and place it on your dedicated cell culture substrate (glass slide, plastic Petri dish, multiwells plate)
  3)  Optional : use a flat surface to make sure the Stencell properly stick to the cell culture substrate
  4)  Immerse the sample in complete cell culture medium and place it in an incubator (37 °C, 5 % CO2)
  5)  Detach your cells according to your standard procedure, centrifuge.
  6)  Remove the bubbles present at the interface Stencell/cell cuture substrate using a pipette.
  7)  Seed the cells and place the sample back in the incubator until cells are spread.
  8)  Fix with PFA 4 % for 12 min, rinse 3 times with PBS.
  9)  Carefully remove the Stencell using tweezers while maintaining one corner with a pipette.
  10)  Stain the cells with standard procedures.
  11)  Optional: mount the glass coverslip on a glass slide.
  12)  Image on a fluorescence microscope.

SpheroRuler beads imaged using SRRF-Stream super-resolution 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, 2023

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

Technical information

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

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

1

Tunable stiffness in a physiological range [1-150] kPa

Use of agarose as a cheap and biocompatible polymer

2

Open access to the reservoir

Possibility to add drugs, and reagents

Easy medium renewal

3

Autoclavable & reusable systems

4

Compatibility with multiple microscopy techniques

Confocal, spinning, super-resolution

Open access for microscope objectives

Use of optical glass coverslip to make cells grow

5

Easy to recover coverslip with cells for subsequent molecular analysis

FACS, qPCR, Western-Blot, Immunofluorescence (possible in situ)

6

Long-term analysis of the cell adaptation to confinement

Up to several days, for time-lapse studies

7

Study of the specific impact of mechanical loads on the biology of cells

Gas permeability of the system allows to get rid of the hypoxia conditions

8

Easy to assemble and disassemble the system

AgarSqueezer – How to use it – A piece of protocol in 3 steps

Resources

Check the most Frequently Asked Questions (FAQ).

Kit description

You can choose among 2 kits:
– The “1 AgarSqueezer kit” contains 1 AgarSqueezer device
– The “2 AgarSqueezers kit” contains 2 AgarSqueezers devices.

In addition to the device, each kit contains also:
– 1x insert to hold up to 2 AgarSqueezers on the microscope stage
– 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)

For users who also need a wafer to mold agarose, we can provide it in 4 different heights:
. 2.5 µm to highly confine cells
. 5 µm to moderately confine cells
. 30 µm not to confine cells (negative control)
. 100 µm for 3D confinement

“In the beginning, in Charlotte’s team, we wanted to analyze the influence of both stiffness and confinement on cells, mimicking highly confined situations such as fibrosis or cancer.
On its side, Veronique’s team was looking for a way to analyze the effect of long-term confinement with the ability to add a drug at any time.

We all wanted to find a device that would also meet the requirements of subsequent classical molecular analysis (easy cell culturing and cells recov​ery, qPCR, western-blot, in situ immunostaining) as well as biophysical image-based analysis (high-resolution microscopy and video-microscopy). But we could not find any.

So all together, we decided to collaborate on the perfect set-up. We rapidly identified agarose as an interesting material to get medium and oxygen renewal, with no drug adsorption. We built several prototypes, improving precise control of the confinement, avoiding destruction of the gels and leakage of the culture medium! And then we came up with the Softconfiner device, that we published and decided to transfer.
Today, we believe that the AgarSqueezer could be of interest for many researchers willing to better understand how mechanics can regulate cell behavior. We hope that researchers from different communities will also find it a useful tool!”

Audrey Prunet, Gilles Simon, Hélène Delanoë-Ayari, Véronique Maguer-Satta and Charlotte Rivière

“When Charlotte showed me for the first time their Softconfiner device, I got highly impressed by its level of maturity. The team of developers had managed to confine cells up to 8 days without affecting the cells viability, but had observed that mechanical confinement affected the cell proliferation of all the tested cell lines. Charlotte and the team were very clear on their motivation to make their Softconfiner easily accessible to the research community. In addition to being a pleasure to work together, it was quite obvious the technology would become a great product.” Esther Graudens
New projects at Idylle

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