Increasing the yield of human mononuclear cells and low serum conditions for in vitro generation of macrophages with M-CSF.
The yield of mononuclear cells extracted from peripheral blood using standard protocols is frequently inadequate when working with material of limited availability. In addition, the regular usage of autologous and fetal calf serum (FCS) to generate human macrophages in vitro may complicate antigen uptake, processing and presentation on HLA molecules. We optimized the yield of mononuclear cells from 34+/-3% to 65+/-5% by collecting both the interface and more than half of the overlayering supernatant, followed by three washes at 4 degrees C. Monocytes from 12 individuals were cultured 1-4 days with 0-100 ng/ml macrophage colony-stimulating factor (M-CSF) at either 1% (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50-100 ng/ml) M-CSF stimulation and low FCS induced 65+/-5% esterase-positive cells in all individuals compared to 52+/-7% without M-CSF (p<0.001). M-CSF increased the mean proportion of esterase-positive cells in both 1% and 5% FCS with a negative interaction found between 5% FCS and M-CSF (p<0.05). All cells were positive for CD14 and HLA class II but cell number did not increase. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.
Plesner
Annette
A
Department of Medicine, University of Washington, Box 357710, Seattle, WA 98195, USA. plesner@interchange.ubc.ca
D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D016173:Macrophage Colony-Stimulating Factor / Q000502:physiology*; D008264:Macrophages / Q000502:physiology*
D016173:Macrophage Colony-Stimulating Factor