We are trying to isolate primary human monocytes from peripheral blood. Following PBMC isolation with Ficoll, we want to enrich monocytes by herence. We culture PBMC with serum-free 1640 for 2-24 hours in 6 well plate and then discard non-adherent cells. The herent cells were cultured with 10% FBS-1640. However,there are so many non-adherent cells and we did not obtain enough cells. We have tried 2, 4, 6, 12 and 24 hours to adhere tightly.
Any suggestions on anything I could try? Could I envelop 6 well plate with poly-L-lysine? We want to differentiated monocytes to foam cell. Could I directly stimulate PMBS without purifing monocytes?
Why do you want to go for the adherence? It's rather old-fashioned, and yields poor purity (and other contaminating PBMCs may then affect monocyte differentiation to macrophages/foam cells). I strongly recommend magnetic bead separation, either via negative or positive selection (positive selection usually resulting in >99.5% purity); there are several kits from multiple manufacturers available on the market.
Anna Ohradanova-Repic I just want to perform Pre-Experiment to preliminarily identify trends. Magnetic bead separation is too expensive for me.
The following is a standard human monocyte isolation protocol. You may want to scale it for plates with a larger surface area. The magic is using 5% HSA and adherence for 1 hr at 37C followed by 3 washes with HBSS. Longer time periods or the use of FBS does not work as well. Some where both Josh and I have published SOPs but would take much time to find.
Isolation of Human Monocytes. MNL were isolated from peripheral blood on lymphocyte separation medium (Organon Teknika, Durham, NC), washed twice in HBSS, and then suspended in RPMI 1640 containing 5% human AB serum. The percentage of monocytes in the MNL layer was assessed by morphology and peroxidase stain, and the cell suspension was adjusted to contain 1 x IO6 monocytes/ml. Into each well of a 96-well flat-bottomed Microtest II plate (Falcon Plastics, Oxnard, CA), 2 x IO5monocytes were added and allowed to adhere for l h at 37°C.Nonadherent cells were removed by 3 washes with HBSS. The purity of the adherent monocyte monolayers was >97% as assessed by India ink ingestion, morphology, and peroxidase staining.
From Activation of Tumoricidal Properties in Monocytes from Cancer Patients following Intravenous Administration of Liposomes Containing Muramyl Tripeptide Phosphatidylethanolamine1
Eugenie S. Kleinerman;
James L. Murray;
John S. Snyder;
Joan E. Cunningham;
Isaiah J. Fidler
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