Could someone kindly explain to me why some protocol do not wash the cells after staining with FITC-Annexin V and PI, to get rid of unbound annexin V/PI ?
I guess the main reason is to reduce the time the cells interact with Annexin V (it may be toxic for cells and after long incubation more and more cells will stain positive). From personal experience I also noticed that many commercially available Annexin V antibodies are very diluted- likely because of annexin V toxicity (for flow cytometry antibodies concentrations should be "saturating", otherwise staining will be highly affected by cell numbers) . So it's important not to exceed recommended cell numbers for single staining. In regards of PI, 7AAD or DAPI ( and other DNA dyes ) - these are DNA binding dyes, they get into the cell if it's membrane is compromised, so there is no much sense in washing cells after staining, unless there are following fixation /permeabilization steps. I usually resuspend cell's pellets in 1x PI or DAPI solution at the very end of "live" (unfixed) cells staining and never wash after that, and always have very consistent staining.
Hi,
I think it´s because of the DNA dyes. Their affinity to DNA is not too high and the washes free them into solution, causing signal loss.
Annexin V has a relatively high affinity to phosphatidylserine so there´s no trouble in washing (like an antibody) as long as there is calcium in solution (binding is calcium dependant).
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