Hello everyone,
In my experiment, I have two conditions: PBMC alone and PBMC co-culture with synoviocytes in 6-well plate for 48h. PBMC were from frozen PBMC, and after thawing , the percentage of alive PBMC was about 70% to 80%. After I recovered cells for cell staining and FACS. The recover protocol was to add 0.5ml of EDTA each well for 10min at incubator and later add 1ml of RPMI medium. After centrifugation at 1800rpm for 10min, suspended PBMC in FACS buffer and then did surface staining as the normal protocol. But why did I always get the low cell viability of PBMC including the unstained PBMC, was about 50%. I dont know whether it resulted from the use of EDTA. But if I did not use EDTA, how can I detach synoviocyts in the co-culture? Because in co-culture system, some PBMC contacted with synoviocytes, it's necessary to detach synoviocytes in order not to lose these PBMC.
Does anyone have experience? Thank you so much!
I attache the FSC-SSC plot in unstained PBMC. I thought the left subset is dead cells, so I chose to gate the right subset. But the percentage was only about 50%. Could anyone have done the similar experiment? Thank you.
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