I am working on stimulation of PBMCs isolated from HIV positive individuals using 10%FBS RPMI and HIV gag p24 (1µg/ml) for three days to investigate some inhibitory receptors (Like PD-1) immunocenescence (CD57) and immunoactivation (CD28) markers on the surface of T cells by flow cytometry. I have seeded 300,000 cells in each well of 24-well cell culture plate and I have not harvested more than 1 million cells pulling up five wells after 3 days. I would like to know if three days is not too much to culture and then how can I generally increase the number of cells.
I usually add 125u/ml of IL-2 although it doesn't effect the experiment even if i dont add IL-2.
Try IL-7 for 2-3 days and Il-7/IL-15 from DAY 3 onwards if you want to drive out the cells a little more. I find IL-2 works but can also cause cellular death in this short time period. IL-7 works for me.
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