how can EDTA (added to trypsine when deattaching cells to remove calcium) affect flow cytometry results? does it bind to surface proteins and hinders Ab-Ag bunding?
EDTA is predominantly used for the chelation of divalent ions, as such it should have minimal direct binding to any proteins. It may effect antibody binding in situations where the structure of the epitope depends heavily on ions such as calcium, otherwise this should have negligible effects. It's going to depend largely on what your antigen is.
Treating your cells with trypsin might have a more serious effect on your staining than the EDTA as it can destroy your antigen.
I can only agree with my the previous answers.
If you are using trypsin to separate cells some epitopes may be destroyed due to digestion. The EDTA will mainly preventclustering by cating divalent ions like calcium. Additionally it can be used for detaching and preventing reattachement of cells. Only if the anitbody is raised against an epitope that requires divalent ions it could be a problem.
However, if you are not sure if your epitope could be affected you can try the following. Trypsinize your cells and cultivate them for 4-12h on a rocking plate to circumvent reattachement and clustering. This should allow the recovering of destroyed epitopes. But this procedure is not working for all cell lines/differentiated cells, esepessially when they like ti form clusters because then shaking can even increase their clustering.
Hope it helps you to solve your problem. Greetings
EDTA should not negatively affect protein detection as it is used to quelate metal ions that play a role in cellular adhesion. Trypsin, however, can degrade your protein of interest so that if it is a low abundant protein it might not be detected in your flow cytometry analysis.
I have experienced this myself. The solution was using a non enzymatic cell dissociation solution. I was told this solutions may cause a lot of cell death, but in colon cancer cell lines I got around 90% of cell survivance.
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