Dear all,

I am trying to isolate PBMCs from human blood. I am doing a gradient centribugation to remove most of the erythrocytes. The leftover erythrocytes I get rid of them using ACK lysis buffer for 3 minutes at RT. All steps I do at RT and very gently. I am using HBSS Ca-/Mg- with BSA 0.5% and Human FcR block from BD as blocking solution, for 20minutes at room temperature. I do the antibody incubation at RT for 30minutes in the dark. When I analyze by FACS the P1 population I get a double granulocyte population that stains positive for classical neutrophils markers (see picture attached). I assume the second population are activated neutrophils. Have anyone have experience this too? Any idea to get more consistent PBMC isolations? Thanks. Miguel