I need to isolate macrophages from PBMC for RNA seq. The macrophages will be isolated 5 hours after activation (with Zymosan,through TLR 2/6). What would be the best method?
And if I isolate them through negative selection, which antibodies should be present in the selection mix?
Hi Michal Levy,
Negative selection is the best way to have a pure (unlabelled) population of monocytes from PBMCs. You could then activate these freshly isloated monocytes to have a macrophage population. I used this kit (link below) from miltneyi for my expts and everything went smoothly.
https://www.miltenyibiotec.com/US-en/products/classical-monocyte-isolation-kit-human.html
Neha
Hello Michal, I found the easiest way to isolate mono/macrophages directly from PBMC, was to lyse rbc -> wash -> incubate the sample, TC plastic dish/flask, in suitable TC media -> 1-2 hours, under TC conditions. Remove/transfer non-adherent cells(lymphocytes). Wash dish/flask, and mono/macrophages recovered with TrypLE.
If you wish the check efficiency of the process -> stain aliquots( before, post non-adherent and post adherent) with appropriate marker eg CD14.
I fortunate to have a cell sorter for RNAseq research -> sorting appropriately labelled cells directly into microtubes.
Best wishes
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