We are having a lot of trouble with CFSE stained K562 cells as almost 97-98% of the stained cells lose CFSE in about an hour spontaneously. We need to use these cells in a NK cell cytotoxicity assay. Has anyone else faced such issues ? Any suggestions will be very helpful. Thanks.
Hi Manjunatha M Venkataswamy . Carboxyfluorescein bleaches readily when exposed to light. Keep labeled cells in covered vials away from light.
Alternatives are rhodamine and Atto dyes that do not bleach as rapidly.
Dear Stefansson,
Thanks for your suggestions. We have been careful not to expose the stained cells to light. We also tried staining the K562 obtained from two different sources. Could it be a problem with the CFSE dye that we are using? Will try using the other dyes you suggested.
Best,
Manju
Hi Manjunatha M Venkataswamy . Are there any oxidation reagents present in your cell labeling protocol? Carboxyfluorescein is very sensitive.
Another possibility: If the cells are alive, then it is a possibility that they are internalizing the labeled cell surface proteins and delivering them to lysosomes. Lysosomes produce H2O2 that bleaches carboxyfluorescein.
Keeping the cells on ice will stop endocytosis.
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