I am doing a NK cytotoxicity assay using the NK-92 cells line and K562 target cells. I prelabel my target cells with CFSE for 20 minutes.
Then I co-culture the NK cells and the labeled target cells for 4 hours and then quantify the dead parentage using PI. Everything sounded good until I noticed all my cells in my low control (K562 only) dead!
I first thought this might be due to the NK-specific medium that I used for the co-culture (Alpha-EMEM + supplements) but even with the conventional RPMI 1640 this happened again!
I use 100% live and healthy K562 cells before the incubation but after 4 hours they all turn dead as quantified by PI!
What is happening? Why are my target cells dying?! Could it be due to CFSE toxicity?!
Thank you in advance!
Hi Malid:
There is little chance for NK kill all target cells in such conditions. To me, it looks like CFSE spillover into your PI channel in your FASC machine (I assume you used PL2 (PE ish) dectetor), then CFSE positive cells will all turned PI positive. To validate this, you can try K562 cells with and without CFSE staining, run on FACS, and look both PL2 and PL4 (APC ish). This can give u some clue.
Regards
Thank you.. yesterday, I thought this may be the problem and so I tried to detect PI in FL3 instead of FL2 and the problem was over! You are right and this was a CFSE spillover into channel 2.
Thank you so much for your information. quite helpful Shiqiu Xiong
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