I am isolating PBMCs with the use of cell protection tubes. After a couple of washes, I get a nice cell pellet. I then add red cell lysing solution to the pellet, but this seems to be lysing my PBMCs. If I don't use the red cell lysing solution, I get fourfold higher PBMC counts. I have to lyse red cells in the PBMC cell pellet, as I am looking at a metabolite that accumulates at much higher concentrations in red cells than in PBMCs. I am isolating PBMCs with the CPT from 4 ml of blood. The original protocol recommended to me was 5ml red cell lysing solution for 10 mins followed by 15 mls PBS wash x 2. I have tried three different red cell lysing solutions (invitrogen, quiagen and a third), with different volumes for the cell pellet (5ml, 1ml, 1 ml + 1ml PBC) for different times (10 min, 5 mins) and with different wash stages after the lysing (two times wash with 15ml PBD, two times wash with 50 ml PBS). But no matter what, the red cell lysing solution still seems to be lysing PBMCs. If I use less time or volume than I have lots of red cells left over.

Does anyone have any insight into why?

Thanks