Yac-1 target cells and NK-cell assays

[SStifter]

Hi everyone,

I'm trying to get an NK-cell assay working and have so far tried an LDH release assay and also by FACS using CFSE/PI. Problem is that I have yet to see any target cell lysis. Is it possible that YAC-1 cells lose their sensitivity to lysis after prolongued periods in culture? I thaw my cells and use them within 3 passages so they don't exactly spend a long time in culture...

I've also tried stimulating the isolated spleen cells with interferon and saw no appreciable difference in cell death. I would go so far as to say I saw not a single dead target cell at an E:T ratio of 100:1, which according to the literature should give me ~10% death in unstimulated cells and >40% in IFN induced NK cells.

Does anyone have any comments? Should I look at my target cells?

Thanks,
Seb