I recently started to differentiate CD4 cells into T reg cells following standard protocol. Plated ~5x10^5 CD4+CD62+FoxP3GFP- naive CD4 (also stained with Invitrogen Live and Dead kit for live cells) per well in 96-well flat bottom plate (total have 4 wells), with plate-bound anti CD3/CD28. Stimulated with TGF-beta and IL-2, and cultures for 5 days. I started to see green cells from day 4, and day 5 there are a lot of green cells. However, after I sorted out all the GFP+ cells, 70% of the live cells are the FoxP3 positive cells.
So I total have ~2 million cells, and end up only getting 2x10^5 cells. And after 5 days of culture, there are ~4x10^5 cells are live cells. Is this normal? Or I am getting too much dead cells during culture?
ps. I did not using feeder cells.
Edited by venuste, 11 March 2010 - 06:02 PM.