Anacyte is the inventor of CellCover, the only reagent that allows parallel storage of proteins, RNA, and DNA in their cellular context. CellCover maintains cellular shape integrity without chemical crosslinking permitting all downstream applications, including single cell RNA sequencing, NGS or protein sequencing. In collaboration with partners and customers, Anacyte Laboratories concentrates on development of integrative multi-analytic tools.


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CellCover

CellCover is the only reagent that allows parallel storage of proteins, RNA, and DNA in their cellular context.

  • Maintain cellular shape integrity without chemical crosslinking.
  • Protect DNA, RNA and proteins, protein expression and gene expression status.
  • Instant stabilization in one fast step.
  • Compatible with staining (in CellCover).
  • Non-toxic, ready to use solution.
  • For use in human and animal cells and solid tissues, including tumors and cultured cells (adherent, suspension, spheroids).
  • Truly representative molecules for genomic analysis, transcriptome analysis, and proteomic analysis.
  • Virtually all downstream applications are compatible with cell fixation using CellCover, including single cell RNA sequencing, NGS, protein sequencing, and more.

Cells Treated with CellCover Maintain Their Native State.

Please scroll down for more information on how to use CellCover.

CellCover stabilizes cells in suspension, biopsies, or tissue without cell lysis, keeping cells close to in vivo morphology. CellCover is compatible with morphological analyses and staining procedures, including immunocytochemistry, immunohistochemistry, and flow cytometry, among many other downstream applications.

A unique and special feature of CellCover is the ability to perform additional molecular analysis on cells that have already been analyzed on a morphological or molecular level: DNA (e.g. PCR), RNA (e.g. RNA seq or single cell RNA-seq) or protein (e.g. protein modification analysis, protein sequencing, or proteomic analysis) can be isolated for subsequent applications after initial immunolabeling, immunohistochemistry, immunocytochemistry, or flow cytometry analyses.

Application of CellCover is simple. Remove cell culture medium, apply CellCover and incubate for two minutes. Then proceed with your standard protocol.

CellCover can be used in the following applications (and many other!):

  • Batch and single cell analysis
  • Flow cytometry / FACS
  • Immunocytochemistry
  • Immunohistochemistry
  •  DNA Sequencing
  • FISH
  • Microarray
  • NGS
  • PCR
  • Protein Sequencing
  • RNA Sequencing (RNA Seq)
  • Single Cell RNA Sequencing
  • Northern Blotting
  • Western Blotting
  • Many more applications

CellCover was developed for fast “one step” stabilization of biomolecules in life science research. CellCover is a non-toxic formulation, a formaldehyde alternative designed to protect the status of DNA, RNA, and protein expression and gene expression in human and animal cells and solid tissues, including tumors and cultured cells (adherent, suspension, spheroids). DNA, RNA, proteins  and cell morphology are all protected.

As soon as cells are exposed to CellCover their metabolic state is frozen without applying low temperatures- the liquid frozen effect. Synthesis and turnover of bio-molecules are stalled. This includes instant disruption of cellular degradation pathways, making cell fixation by CellCover very fast. Chemical degradation is inhibited with CellCover as well, leading to exceptional protein stabilization and protein fixation, as well as RNA fixation and DNA fixation. CellCover acts as an DNA and RNA stabilizer and shield, which is beneficial e. g. for analysis by flow cytometry or in NGS or RNA seq approaches. CellCover is a key initial step for successful experiments in genome analysis, transcriptome analysis, and proteomic analysis.

Cells grown on almost any substrate can be treated with CellCover by liquid freezing to maintain in vivo morphology without chemical crosslinking of biomolecules during cell fixation.

Cell morphology is preserved, allowing for accurate visualization of molecule location. DNA, RNA, and proteins are also stabilized and preserved when performing cell fixation with CellCover, allowing for effective downstream experiments in genomics, transcriptome analysis, and proteomics.

High RIN Values

With CellCover performing RNA fixation and acting as an RNA stabilizer, RNA degradation is inhibited, leading to great results in RNA sequencing or single cell RNA seq or single cell sequencing experiments. Material destined for a biobank is kept without altering expression due to environmentally induced gene expression changes. Even integrity of high molecular weight RNA is protected (e.g. precursor rRNA, which are normally degraded after applying commonly used standard procedures) – and there is no need to hurry! You can store your sample in CellCover at 4°C and isolate RNA later.

Protein expression analysis has no limits. Study protein modification and proteomics with CellCover taking care of protein fixation and protein stabilization. CellCover is a safe formaldehyde alternative fixation, with formalin sensitive epitopes readily accessible for antibodies in immunohistochemistry experiments.

1.1: Standard RNA Integrity Number (RIN) Analysis of RNA

1.2: Cultured cells (SKMel-28) stored for up to 12 days in CellCover. RNA was isolated and its integrity was analyzed with standard procedures. Results show a stable RIN value of 10 from first till last day of the time course. High RIN is important for many gene expression and transcriptome analysis experiments, including batch RNA seq or single cell RNA-seq / single cell RNA seq.

A unique and special feature of CellCover liquid freezing is that you can perform additional molecular analysis on cells already analysed on morphological or molecular level: DNA (for e.g. PCR), RNA (for e.g RNA seq) or protein (for e.g protein modification analysis) can be isolated for subsequent applications after initial IHC, ICC or flow cytometry analyses.

These unique properties now enable new types of analyses of cellular functions. With CellCover liquid freezing, subpopulations of cells can be separated according to known properties by flow cytometry and subsequently analysed for gene expression profiles on RNA or on protein-level. Thus, with CellCover liquid freezing it is easy to link transcriptome and proteome of a given cell population.

(Left: CK Pan, Right: CD31)

Immunohistochemistry

CellCover stabilizes cells in suspension, biopsies or tissue. CellCover is compatible with morphological analyses and staining procedures, with immunocytochemistry and flow cytometry.

(Initial protein analysis & RNA isolation)

Double Analyses

Cells treated with CellCover maintain morphology for several days. Epitopes are protected and can be stained later. Highly intact RNA can be isolated from cells in which protein has already been stained by immunolabeling. Quality of RNA after CellCover protection: Cells have been immunostained for protein, RNA was isolated subsequently.

(Left: Formalin, Right: CellCover)

Immunohistochemistry

CellCover liquid freeze effect allows visualization of formalin sensitive epitopes like Vimentin, enabling new insights in cells structure and architecture.

In comparison to commonly used molecular biological methods, CellCover adds following new analytical tools to bio-molecular research:

 

  • Stabilize RNA and proteins simultaneously for several days, without doubt of molecule integrity
  • Cells maintain close to in vivo morphology without chemical crosslinking of biomolecules allowing straight forward RNA isolation.
  • No freezing necessary
  • Reliable analysis HMW-RNA and low stability enzymes even after day of storage
  • Apply immunohistochemistry on formalin sensitive epitopes
  • Apply in situ hybridization for analysis of gene expression
  • Store cells conveniently for several days without change of protein or RNA expression profile or change in cells morphology as found with methanol
  • Isolate subpopulations by flow cytometry based on protein labelling and perform subsequent transcriptome and/or proteome (MALDI) analyses.
  • Next generation sequencing and single cell RNA seq.
  • Reliable sample collection for biobank deposits.
  • “CellCover preserved nucleic acids of cells underwent immunofluorescence staining and imaging. Cell fixation with CellCover preserved more nucleic acids compared to formaldehyde and methanol fixation for our microscopy experiments.”

D. Wu, Diagnostics Company in US, Cancer Immunology

  • “We wanted to collect clear RNA from Neutrophil which has low RNA and short survival. After collecting Neutrophils, we fixed with cellcover immdiatly. It was good for collecting quality of RNA because it can use for RNA sequences.”

Jun Takai, Tohoku Medical and Pharmaceutical University, Division of Medical Biochemistry

  • “I prefer CellCover to other fixatives such as PFA as it does not require a wash to remove. We are also able to extract more RNA per cell when compared to other fixation methods.”

Josh, Diagnostics Company in US, Oncology

  • “Since the cells are fixed, we do not always have to perform quickly for staining with antibody, FACS, and RNA extraction after collecting cells. In other words, it is good for starting our experiment at our convenience.”

Yuki Sato, Kyushu University – Graduate School of Medical Sciences, Department of Anatomy and Cell Biology

  • “Mostly I used CellCover for sorting of brain microgrial cell and neuron. Regarding CellCover, recently one paper came out from our laboratory where I used CellCover.”

Choudhry Emamussalehin, Ehime University –  Graduate School of Medicine, Molecular and Cellular Physiology

Trypsinization

Avoid Manipulation Induced Change of Gene Expression.

Optimize Your Trypsinization Protocol by Use of CellCover.

As soon as cells experienced changes in their environment they adapt signal transduction and gene expression to it. Thus, upon temperature change and/or cell culture medium removal and trypsinization, cells adapt transcriptome and proteome very rapidly. Keeping the true cell. The rapid fixation of RNA and proteins is very important for expression patterns to remain unchanged. Such rapid fixation is often at the expense of other factors such as molecule crosslinking by PFA or cell lysis by high-salt reagents that stabilize RNA. Not so with CellCover, which instantly fixes and stabilizes cells in their true state of RNA and protein expression

Have you ever fixed your cells directly in their culture flask and then performed trypsinization for further experiments?

Probably not. But with CellCover you can, saving materials, time, and money.
Most importantly, with CellCover fixation followed by trypsinization, excellent sample quality is maintained with no time given for changes in the sample to occur.

There is even more to it.

Not only perfectly fixed RNA is retained after trypsinization, but typical cellular morphology is also maintained and the antigens are readily accessible for a wide variety of applications (IF, IHC etc.).

Sticking to scheduled time points when finishing and working up cell culture experiments

Special protocol for adherent cultured cells:
  1. Remove cell culture media
  2. Add appropriate volume of CellCover and incubate for 2-3 min at room temperature
  3. Remove CellCover
  4. Add trypsin solution (e.g. Trypsin/EDTA 0,25% for adherent cells) and incubate for 5 min at 37°C (or until single cells detached)
  5. Collect cells by centrifugation
  6. Resuspend cells in CellCover and store at 4°C until needed or proceed with your (downstream) application(s)

Application of CellCover is simple:

Remove cell culture medium, apply CellCover and incubate for two minutes. Then proceed with your standard protocol.

  • Batch and single cell analysis
  • Flow cytometry / FACS
  • Immunocytochemistry
  • Immunohistochemistry
  • FISH
  • Microarray
  • NGS
  • PCR
  • RNA Sequencing
  • Northern Blotting
  • Western Blotting
  • Many more applications

Protocols

Basic Protocol 1

Basic protocol for adherent cultured cells:

  1. Seed and grow cells on chamber slides
  2. Remove medium
  3. Wash cells 1x with PBS or CellCover
  4. Place slide in CellCover and store at 4°C until use
  5. Proceed to staining protocol according to experimental design, e.g. immunostaining (If RNA is to be isolated for downstream application, you can stain cells by using CellCover)

Basic Protocol 2

Basic immuno-staining protocol for suspension cells:

  1. Harvest cells by centrifugation
  2. Remove supernatant
  3. Flick tube to suspend cells in residual medium
  4. Add CellCover (5 to 10x volumes) and store at 4°C until use
  5. Pellet cells and remove SN
    (Some cells might need higher centrifugation speed for pelleting.)
  6. Proceed to standard staining protocols, e.g. immunostaining (If RNA is to be isolated for downstream application, you can stain cells by using CellCover as antibody diluent and washing buffer

Basic Protocol 3

Basic protocol for tissue specimens:

  1. Cut tissue into approximately 5 mm pieces
  2. CellCover does not penetrate tight junctions, thus encapsulated organs must be open across the largest diameter.
  3. Place tissue in  CellCover (at least 10x volumes) and store at 4°C until use
  4. Change CellCover at least once after 4-24 hours
  5. Proceed according to experimental design e.g. RNA isolation

Can I store my purified RNA in CellCover?

Yes, you can. Storage of RNA in CellCoverprotects your RNA sufficiently for most subsequent purposes.

How shall I store my purified RNA in CellCover?

This really depends on when do you need it. After purification you usually have it on ice. If you need it for e. g. a Northern analysis within the next two days, keep it on ice (best in a cold room for the ice lasts longer). If you are not sure when you start your subsequent analysis, freeze it at minus 20°C. However, if you want proper maintenance for several month consider storage at -80°C

My purified RNA is partly degraded, what might have been wrong?

Degradation, usually from the 5´end, is a common problem when using purification kits. We recommend to strictly adhere to instructions of the manufacturer of the kit and to work as cold as possible (above 0°C). After precipitation, we recommend dissolving purified RNA in ice-cold CellCover.

Does my purified RNA degrade in CellCover?

RNA protection by CellCoveris almost as effective as deep freezing and sufficient for most purposes (e.g. no change in RIN after 24 h on ice).

What are typical RIN values if using CellCover?

RIN values using CellCover are generally among the best you can get. You can expect RIN values of 10 even after storing cells for days in a fridge. However, many variables influence the individual experiment, e.g. RIN value increases the more experienced the researcher is, is dependent on the kit used for purification and might differ from cell type to cell type.

In tissue, how long does it take until my RNA is protected with CellCover?

CellCover works on to ends, chemical stabilisation and protection against enzymatic degradation. Time to fully protect RNA increases in the following order: non-adherent cells- adherent cells – spheroids – tissue. As a guideline: the more cells, the longer it takes. Usually storing a 5 mm tissue block sample on ice o/n is sufficient. Store spleen or central nervous system tissue for at least 24 h on ice.

Can I freeze my tissue samples in CellCover?

You can freeze your sample in CellCover, if protein in your sample is of interest for you. However, you should leave your sample on ice o/n for some time before freezing.

Can I analyse my cells in CellCover with MALDI?

Yes, you can. Follow your lab protocol. Ingredients of CellCover do not interfere with macromolecular analyses.

Does CellCover protect HMW-RNA?

CellCover protects5´prime ends of RNA , so you can analyse large RNA species more easily. Some 20 kbases should not be a problem.

Is there something important to know when isolating RNA from tissue in CellCover?

Any manipulation might affect your RNA. Cell lysis is one of the most critical steps for it must be fast and efficient. Cells can be resuspended in a small volume CellCover before adding Lysis Buffer. Tissue should be minced on ice or grind under liquid nitrogen before adding Lysis Buffer.


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