Differentiation to M1/M2 macrophages GM-CSF/M-CSF

Contributed by Martin Fitzpatrick, University of Birmingham, United Kingdom

GM-CSF/M-CSF differentiation of CD14+ PBMCs to M1/M2 macrophage subsets.

method/12/750px-Macrophage.jpg

Requirements

GM-CSF stock at 100ug/ml in PBS

M-CSF stock at 100ug/ml in PBS

Method

  • 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.

References

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