MDA-MB-231 human breast carcinoma cells expressing GFP (MDA231) were kindly provided by Dr. Frank Gertler, MIT. PC3 human prostate carcinoma cells (PC3), MDA-MB-435S human melanoma cells (MDA435), and Raw 264.7 mouse macrophages (Raw) were obtained from American Type Culture Collection. MDA231, MDA435, and Raw cells were cultured in DMEM. PC3 were cultured in RPMI. All media were supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin/streptomycin. Cell lines were authenticated using Short Tandem Repeat profiling (Promega).
To generate primary bone marrow-derived macrophages (BMDM), bone marrow cells were first isolated from the femurs of C57BL/6 mice. These cells were then differentiated in RPMI supplemented with 10% FBS, 1% HEPES, 40 ng/mL MCSF (Peprotech) and 50 µM β-Mercaptoethanol for 7 days to produce BMDM. Primary human monocyte-derived macrophages (MDMΦ) were generated from monocytes isolated from whole blood (Research Blood Component) using a Ficoll-Paque gradient and the EasySep™ Monocyte Enrichment Kit (StemCell Tech.). These cells were cultured with IMDM supplemented with 2% L-glutamine and AB serum for 7 days to generate MDMΦ. All cells were cultured in a humidified incubator at 5% CO2 and 37 °C.
To generate primary bone marrow-derived macrophages (BMDM), bone marrow cells were first isolated from the femurs of C57BL/6 mice. These cells were then differentiated in RPMI supplemented with 10% FBS, 1% HEPES, 40 ng/mL MCSF (Peprotech) and 50 µM β-Mercaptoethanol for 7 days to produce BMDM. Primary human monocyte-derived macrophages (MDMΦ) were generated from monocytes isolated from whole blood (Research Blood Component) using a Ficoll-Paque gradient and the EasySep™ Monocyte Enrichment Kit (StemCell Tech.). These cells were cultured with IMDM supplemented with 2% L-glutamine and AB serum for 7 days to generate MDMΦ. All cells were cultured in a humidified incubator at 5% CO2 and 37 °C.