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Q1 – Can CellCover be used with bacterial cells or plant cells?
No. CellCover has only been qualified with mammalian cells. It is likely to work with any cell type without a cell wall.
Q2 – Can CellCover work with Organoids and Spheroids?
Yes. Depending upon the size, we would normally recommend the following protocol for small cell clusters (less than 1 mm in diameter):
- Remove culture media
- Add CellCover (1 mL to 2 mL)
- Remove CellCover after 2 minutes
- Add fresh CellCover (1 mL to 2 mL)
- Use immediately or store at 4°C until needed
Q3 – How long can cells and tissues be stored in CellCover before downstream analyses are performed?
Cells preserved with CellCover can be used immediately. Or these cells can be stored at 4°C for a 1 week without noticeable degradation of biomolecules or changes to cell morphology. Longer storage is possible, but should be empirically determined.
Q4 – Will CellCover inhibit the ability of GFP- and RFP-fusion proteins, or mask their epitopes after treatment?
No. Fluorescence of GFP and RFP is not affected by CellCover. And because CellCover does not fix by making covalent bonds, epitopes for for binding with antibodies are typically preserved after treatment.
Q5 – Will enzymes treated with CellCover be able to maintain their native enzymatic activity?
Maybe. This depends on the enzyme. Because CellCover does not fix by making covalent bonds, at least some enzymes will regain activity if placed back into a physiologically-buffered environment.
Q6 – Can cells be cultured following treatment with CellCover?
No. Cells will not survive treatment with this reagent. CellCover is a unique cell fixative that works quickly on a broad range of biomolecules without chemical cross-linking.
Q7 – What is the viscosity of CellCover reagent?
The viscosity of CellCover is comparable to water. So CellCover is easy to pipet, easy to apply to your sample and will have little loss by getting caught in the pipet tip.
Q8 – Is CellCover compatible with co-immunoprecipitation experiments?
Yes. Generally CellCover is compatible with immunoprecipitation because it does not covalently crosslink molecules. We recommend that lysis be done with RIPA buffer, however RIPA by itself might cause some protein complexes to dissociate so this must be empirically determined.
Q9 – Will CellCover lyse red blood cells?
There is no specific lysing agent in CellCover. Red blood cells that are treated will round up and get cleared, however, the plasma membrane should remain intact.
Q10 – Regarding immunocytochemistry (ICC), does CellCover permeabilize the cells, and thus provide access to the cell interior for antibodies?
No, CellCover does not contain any detergent. Please add detergent to allow antibodies to have access to intracellular antigens during ICC staining protocol.
Q11 – Is it possible to perform co-immunoprecipitation, or pull-down, protein-RNA complexes from cells or tissues fixed with CellCover?
Yes it is possible. However, because there are many factors affecting such interactions, a negative result doesn’t necessarily mean that it doesn’t exist.