Mouse m csf

Mice were sacrificed and hind legs were removed and placed in conical tubes containing 5% FCS DMEM media (Gibco). Muscle was removed from tibia and femur bones and the ends of bones were cut off. Using a 271/2 gauge needle, 10 mL of sterile DMEM media supplemented with 10% FBS, pen/strep, and L-glutamine was passed through the bone. Cells were pelleted and plated with mouse MCSF (50 ng/mL) (BioLegend). Following 4 days of incubation, supernatant was removed and plated in a new dish. Cells were again incubated in media with mouse MCSF for 3 more days. Dishes were then washed 3 times with sterile PBS to remove unadhered cells. BMDMs were gently scraped off the dish, pelleted, counted, and seeded in new plates for experimentation. In the case of experiments with calcium-free medium, immediately prior to experimentation, cells were washed three times with PBS, before adding calcium-free DMEM (Gibco) with pen/strep and L-glutamine. Controls for calcium-free medium experiment were placed in calcium-sufficient DMEM with pen/strep and L-glutamine. In some experiments, cells were treated with DMOG (200 μM), GsmT×4 (5 μM), Brefeldin-A (3 μg/mL), recombinant EDN1 (conc), Bosentan (10 μM), Cyclosporine A (10 μM), SR 11302 (10 μM), Echinomycin (5 nm), Bapta-AM (10 μM), Chloroquine (100 μM) or MG132 (50 μM)

Validated Pgam5 gene-targeted ES cells (B6 background: JM8.N4) were purchased from the European Mouse Mutant Cell Repository and chimeric mice were derived by microinjection of ES cells into blastocyst donors. Pgam5 KO mice were generated by breeding chimeric animals (Laboratory Animal Service Program of NCI-Frederick). Pgam5 WT and KO mice were bred and maintained in NIH animal facility at SPF (Specific Pathogen Free) conditions. Mouse experiments were carried out in accordance with NIAID Animal Care and Use Committee guidelines under an approved protocol. 12-month old female mice were used in all the animal study unless specifically indicated. MEF cells were prepared from day 12.5 embryos and cultured in DMEM/F12 full media (FM) (FM: 10% fetal bovine serum, 4 mM L-glutamine, 100 IU ml⁻¹ penicillin, and 100 mg ml⁻¹streptomycin) plus non-essential amino acids. Bone marrow cells were cultured in RPMI FM plus 20 ng ml⁻¹ mouse M-CSF (Biolegend) for 7 days to induce macrophage differentiation. Parl WT and KO MEFs are from Dr. Bart De Strooper (Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology (VIB4) and K.U. Leuven, Leuven, Belgium).

Mouse BMDMs were differentiated from monocytes in the bone marrow. Briefly, femurs and tibias were dissected from both legs of a C57BL/6 mouse, and one end of each bone was cut open with a pair of scissors. Bone marrow was obtained by centrifuging bones with open ends at 2,000g for 30 s. The collected bone marrow was resuspended and cultured in DMEM supplemented with 10% FBS, 100 U ml⁻¹ pen–strep, 2 mM l-glutamine and 100 ng ml⁻¹ mouse M-CSF (BioLegend) for 7 d at 37 °C with 5% CO₂ to obtain mouse macrophages. Medium was refreshed on day 4, and before signaling assays, mouse macrophages were starved in growth medium overnight with 2% FBS.