GM-CSF/M-CSF differentiation of CD14+ PBMCs to M1/M2 macrophage subsets.
GM-CSF stock at 100ug/ml in PBS M-CSF stock at 100ug/ml in PBS
Prepare differentiation media for target differentiation subset, in 50ml total volume RPMI 1640 + 1% GPS + 10% Hi FCS.
M1 macrophages add 5ul GM-CSF
M2 macrophages add 5ul M-CSF
Transfer cells to 50ml tube and pellet by centrifugation 1200rpm 6 minutes
Resuspend cells in media for target subset at 1x106 cells/ml. e.g.
To plate in 6 well plate, cells should be seeded at 3x106 cells/well in 2ml total volume.
Plate out cells or transfer to 25cm2 flask to culture. Incubate for 2 days.
Replace 2ml media by tilting plate carefully, and removing supernatant at edge. Note that cells will not have fully adhered at this timepoint. Incubate further 2 days.
Cells should have fully differentiated. Replace 2ml media again for experiment.
A. J. Fleetwood, H. Dinh, A. D. Cook, P. J Hertzog, J. A. Hamilton GM-CSF- and M-CSF-dependent macrophage phenotypes display differential dependence on Type I interferon signaling Journal of Leukocyte Biology (2009) 10.1189/jlb.1108702