I stained one cryovial PBMCs according to the CFSE protocol (invitrogen C34570). One day before staining, I thawed the cells and rested them in media (ingredients is below ) for 24 hours. On the second day, I removed media and washed cells twice with DPBS then stained the cells with CFSE, cultured them in a 96-well V bottom plate, and stimulated them with 1% PHA (GibcoTM). After 5 days, I excluded dead cells by zombie NIR (Biolegeng cat. 423105) and read them by flow cytometry, but a lot of cells were dead after 5 days in the well that PBMCs stained with CFSE but not in the well where the cells were not stained with CFSE. I think CFSE staining affects the cells, and the cells are dead after 5 days. I want to know how I can solve this problem?
( PBMC media composition: 1X 2-mercaptoethanol (1000X Gibco Ref: 21985-023) 1X MEM non-essential amino acids (100X Gibco Ref: 1140-050) 1X Sodium pyruvate (100 mM Gibco Ref: 11360-070) 1X penicillin/streptomycin (100X Gibco Ref: 10378-016) 10% inactivated FBS RPMI 1640 + L-glutamine (Gibco Ref: 11875-093))
Also, I repeated the test in a 24-well plate and washed cells once instead of twice before CFSE staining. I also added DMSO in parallel with CFSE staining to check the cytotoxicity of DMSO on cells, but unfortunately, after 5 days, only PBMCs that stained with CFSE were dead (5-10 % live cells), but not PBMCs without CFSE staining (50-70% live cells) or PBMCs with DMSO (36% live cells) . I don’t know what is wrong with CFSE staining because it seems to have a cytotoxic effect on cells, and the cells will be dead after 5 days.
Is it possible I face this problem because I used PBMCs that were frozen one year ago?
Or is an experimental compound resulting in cytotoxicity?
Dear Neda K. Dezfuli
I briefly wanted to reach out concerning your inquiry. Firstly, CFSE is absolutely cytotoxic as is it binds the amino acid lysine of intracellular proteins via its succinimidyl groups, that will lead to cross linking of proteins and oftentimes cell death. So in that respect, it's not surprising that your cell viability goes down.
The only way that I found to circumvent that was titrating down the CFSE concentration used for staining and using FBS fortified PBS for all staining procedures. Please find attached, my tried and tested CFSE staining protocol (optimized for mouse cells, but easily applicable for PBMCs).
Please also check for more details:
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
Article MYSM1-dependent checkpoints in B cell lineage differentiatio...
Article Ubiquitin Specific Protease 21 Is Dispensable for Normal Dev...
https://brcf.medicine.umich.edu/cores/flow-cytometry/training-education/lessons/lesson-seven-cfse/
Article Use of the Intracellular Fluorescent Dye CFSE to Monitor Lym...
All the best & good luck with your experiments,
Michael
Michael Förster
Hi
Thank you so much for the protocol. I will try it.
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