Hi, I am doing the culture PBMCs from healthy humans in which B cells are stimulated by anti-Ig and TLR7 ligand. Very oddly, on day2, I see dramatic decrease in the ratio of B cells compared to unstimulated control in flow cytometry. The B cell fraction often restore after 4 or 5 days, but I want to sort B cells on day2.

One example. B cells make up 2.5% in the unstimulated sample, and it decreased to 0.3% in the stimulated one.

I tried to gate B cells both by CD19 and CD20, but the result didn't differ, so I guess it's not due to internalization of those markers.

I want to know if it is common that B cell ratio decrease temporarily after stimulation.

My protocol is below.

PBMCs are seeded on 96 round well-plate by 2 x 10^5 in 200uL per well.

The cells are stimulated by 10 ug/mL of anti-Ig (Jackson), 1 ug/mL R848 and 20 ng/mL(BAFF, peprotech)

*When I tried 5 x 10^5 per well, it decreased with lower degree, so I am guessing cell number matters.

Please give me any advice.