I am trying to isolate naive CD4 lymphocytes from mice spleen, using a cocktail of biotinylated abs and magnetic streptavidin micro-beads, all through AutoMacs columns using the DepleteS program. We obtained a similar number of CD4 cells in my negative fraction (near 25- 30%) and a very low number (1.106 cells). I have tried to test different antibody concentrations and incubation times but nothing works. Could you give me some advice to improve the purity?
P.S. Negative selection is necessary to obtain untouched naive CD4 lymphocytes.