Sometimes when I sort CD8+ cells first from PBMCs and then CD4+ cells, I get a very good purity with the CD8+ beads and a lousy purity with the CD4 beads.
I try to keep the beads, buffer, even the magnet and column on ice before I start. I work fast and use 2 different magnets (kept on ice before I need them). But yet, sometimes, the CD4 population is less pure.
If i sort only CD4 cells, then I get good purities, but I need to do both populations in the same time.
Im not really sure if Im doing a mistake and where that mistake could be or if my beads are contaminated or one of the magnets is not good....
Did anyone have a similar issue? Or do you have any suggestions on how to make sure everything is ok?
PS: i do preseparation filtering. And sometimes i filter for clumps after the CD8 sort and before the CD4 sort, because Im paranoid (sometimes it works nicely and I get >96% purity, but sometimes it doesnt).
Hi Georgina,
we used microbeads for the isolation of CD4+ T cells from spleen single cell suspension (and therefore less purified material), but never faced those problems.
You might want to consider the following points:
- When working with anticoagulated peripheral blood or buffy coat, peripheral blood mononuclear cells (PBMCs) should be isolated by density gradient centrifugation (Ficoll)
- When working with lysed blood, prepare a single-cell suspension using standard methods.
- Dead cells may bind non-specifically to Microbeads, but can be removed using density gradient centrifugation or Dead Cell Removal Kits.
- For the isolation of highly pure T helper (CD4) cells, the depletion of monocytes (CD14) can be neccessary prior to CD4+ T cell enrichment.
- When working with higher cell numbers than stated by the manufacturer of the beads (usually 1x10^7 cells), scale up all reagent volumes and total volumes accordingly.
- Working on ice (as necessary) may require increased incubation times, therefore a prolonged incubation time (15-20 min) with CD4 beads might result in higher purity.
Best of luck!