Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood and subsequent culture of macrophage cells.

This protocol uses centrifugation and ficoll gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs) from whole blood. The Miltenyi Biotec MACS magnetic cell separation system is then used to isolate the CD14+ monocyte fraction from the PBMCs. Subsequent culture with colony stimulating factor 1(CSF-1) differentiates the CD14+ monocytes into macrophages.

This protocol assumes that you are starting with fresh blood mixed with an anticoagulating agent such as ACD, ideally around 300mls in multiple 50ml syringes,   provided for you by a qualified phlebotomist.