I am trying to isolate monocytes to evaluate immunological markers by ELISA and Flow Cytometry. PBMCs are obtained from peripheral blood of healthy donors in heparin tubes, separated by a Ficoll density gradient. Subsequently, the monocyte separation step is carried out by adhesion to the plastic plate for 2 hours in RPMI supplemented with 10% FBS. The supernatant containing lymphocytes is removed. I wash the plate with repeated strong jets of PBS to remove adherent cells from the plate and finish collecting with a cell scraper. I do not use enzymes to remove adherent cells to avoid immune activation.
However, when analyzing the cell population, I find a very low number of CD14+ cells, and a large population of CD3+ cells (lymphocytes).
How to improve this protocol? What are other ways to enrich my sample with monocytes? Is cell sorting an option?
Hi,
I used to use CD14+ selection beads (Miltenyi Biotec) and I got high purity of cells.
Howeve, if you need untouched cells for Flow, you can always do negative selection for classical monocytes Using MACS technology.
Good luck!
Hello Lisandra, We certainly found that a 2->4 hours incubation on plastic, after isolation on Lymphoprep, was the most efficient way to isolate monocytes from human peripheral blood - 3 12% PBMC. However, recovery number and function, was significantly affected by the age of the blood - after 36 hours adherence low and we had difficulty transforming adherent cells(CD14 verified by flow cytometry) to DCs. We found significantly poorer return from bead isolation and greater cost. best wishes
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