Recombinant murine macrophage colony stimulating factor

Both the femurs and tibiae were collected, and the muscle attachments were carefully removed. Intact bones were soaked in 70% (v/v) ethanol for 1 minute for disinfection and then washed with PBS. Next, both ends of the bones were cut with scissors, and the marrow was flushed with Roswell Park Memorial Institute (RPMI) 1640 medium using a 1-mL syringe with a 26-G needle. Clusters within the marrow suspension were disintegrated by vigorous pipetting. After one wash (490× g, 5 minutes) in RPMI 1640 medium, the red blood cells were depleted with 0.83 M NH₄Cl buffer (Sigma-Aldrich). Bone marrow cells (2×10⁶ cells) were collected and cultured in 100-mm Petri dishes containing 10 mL of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 IU mL⁻¹ penicillin, 50 mg mL⁻¹ streptomycin, and 20 ng mL⁻¹ mouse recombinant granulocyte macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). Bone marrow cells (5×10⁵ cells) were collected and cultured in 100-mm Petri dishes containing 10 mL DMEM supplemented with 20% heat-inactivated fetal bovine serum, 50 IU mL⁻¹ penicillin, 50 mg mL⁻¹ streptomycin, and 20 ng mL⁻¹ recombinant murine macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA). After 7 days, nonadherent and loosely adherent cells (BMDCs) or adherent cells (BMDMs) were harvested, washed, and used for in vitro experiments.

To generate peritoneal macrophages, 10- to 12-week-old mice were injected intraperitoneally with 1 mL 3.5% Brewer’s thioglycollate (Sigma-Aldrich). After 3 days, peritoneal macrophages were isolated from peritonitis exudates by peritoneal lavage with 5 mL ice-cold RPMI 1640 medium (Gibco, Waltham, MA). The peritoneal macrophages were allowed to rest overnight in RPMI 1640 supplemented with 10% FBS at 37ºC in a 5% CO₂ incubator before the start of experiments.
To generate BMDMs, tibias and femurs from 6- to 8-week-old mice were harvested. Bone marrows were flushed and passed through 70-μm mesh, resuspended in Dulbecco’s modified Eagle medium (Gibco), and overlaid on Ficoll-Paque Plus (GE Healthcare). The mixtures were centrifuged at 1800 rpm for 20 minutes at 22ºC. BMDMs from the interface of the red Dulbecco’s modified Eagle medium layer and the colorless clear layer were collected and cultured in high-glucose Dulbecco’s modified Eagle medium supplemented with 10% FBS, 2 mmol/L L-glutamine (Gibco), 0.5% 2-mercaptoethanol (Gibco), and 10 ng/mL recombinant murine macrophage colony stimulating factor (Peprotech, Cranbury, NJ) for 7 days before experiments.