Hello everyone,
I'm having trouble detaching peripheral blood mononuclear cells (PBMCs) we got from ATCC out of a TC-treated plate and even a Greiner suspension plate.
I need to do an enrichment protocol for T cells and first I wanted to thaw these cells in a flask and then passage them into smaller batches to try my enrichment protocol.
My T cells are supposed to flow and interact with the magnetic beads I will use for T cell activation, so I need them to be free flowing (ideally).
But the issue is, they don't detach from plastic surface even I used 0.25% trypsin-EDTA, not even after 30 minutes.
Suspension plate is better, but I still saw a few cells that attached to the surface. My trypsin works well on other cells. How do you normally detach PBMCs?
Hi Bora,
Just to clarify, PBMCs consist of monocytes/macrophages, T cells, B cells, and NK cells. The cells attaching to the plate are most likely from the monocyte/macrophage population. Typically, when PBMCs are plated in a plastic container, monocytes/macrophages adhere within a couple of hours (about 2 hours in my experience), while lymphocytes (T cells, B cells, and NK cells) remain in suspension.
There should not be any need to detach the adhered cell if it is the T cell that you want. Moreover, the monocyte/macrophage in PBMCs is less viable after the freeze and thaw, so I would assume that the majority of the cells that you have are already a lymphocyte fraction.
Unless you need the attached monocyte/macrophage fraction to be used as a feeder or antigen-presenting cell during T cell activation, I would not try to detach them because they are extremely hard to detach. Trypsin is not working very well with them; cell scrapers usually tear them apart if you are not careful. I used a high concentration of cold EDTA in PBS (I could not remember the exact concentration anymore), and I have to incubate for 10 mins, twice, to detach them.
Best,
Niphat
Hello Bora,
If you are trying to detach monocytes/macrophage try PBS with 2mM EDTA and 10% FBS, it helps detaching the cells from he plate and also prevent them from sticking to the tube.
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