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Bone marrow-derived macrophage (BMDM) isolation and culture

FS Filipa C. Simões
TC Thomas J. Cahill
AK Amy Kenyon
DG Daria Gavriouchkina
JV Joaquim M. Vieira
XS Xin Sun
DP Daniela Pezzolla
CR Christophe Ravaud
EM Eva Masmanian
MW Michael Weinberger
SM Sarah Mayes
ML Madeleine E. Lemieux
DB Damien N. Barnette
MG Mala Gunadasa-Rohling
RW Ruth M. Williams
DG David R. Greaves
LT Le A. Trinh
SF Scott E. Fraser
SD Sarah L. Dallas
RC Robin P. Choudhury
TS Tatjana Sauka-Spengler
PR Paul R. Riley
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Bone marrow cells were isolated from femurs and tibias of 3–5 months old GFPtpz-collagen transgenic mice. Cells were plated on 100 mm non-tissue culture treated petri dish (Thermo Fisher, UK) and maintained in macrophage differentiation media (DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 2% penicillin/streptomycin and 20% supernatant from L929 cells as a source of macrophage colony-stimulating factor). On day 7 post isolation, BMDMs were positively selected by trypsinization and subsequent wash-out of non-adherent cells. After positive selection, BMDMs were maintained in macrophage-growing media (DMEM supplemented with 10% FBS, 2 mM L-glutamine and 2% penicillin/streptomycin) for 1–2 days before being plated for imaging.

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