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Bone marrow-derived macrophage (BMDM) isolation and culture

NT Nuria Tubau-Juni
JB Josep Bassaganya-Riera
AL Andrew Leber
VZ Victoria Zoccoli-Rodriguez
BK Barbara Kronsteiner
MV Monica Viladomiu
VA Vida Abedi
CP Casandra W. Philipson
RH Raquel Hontecillas
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Bone marrow-derived macrophages (BMDM) were isolated as previously described60. Briefly, bone marrow (BM) was flushed out from sterilized femur and tibia of euthanized mice. Osmotic lysis was used to remove red blood cells. Samples were differentiated in cRPMI (RPMI 1640, 10% Fetal bovine serum, 2.5% Hepes, 1% Sodium pyruvate, 1% l-glutamine, 1% Penicillin/Streptomycin and 50 uM β-mercaptoethanol) supplemented with 25 ng/mL of recombinant mouse macrophage colony-stimulating factor (M-CSF) at 37 °C, 5% CO2 and 95% humidity. At day 3 fresh M-CSF-supplemented media was added. On day 6, BMDM were harvested. Cells were re-suspended in cRPMI and left to adhere overnight at 37 °C, 5% CO2 and 95% humidity.

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