Calcium free dmem

Four to six hours after plating, experimental manipulations were carried out by changing culture medium. Calcium influx was increased by depolarizing with elevated extracellular K⁺ - 25 mM (25K) or 50 mM (50K) - as previously described ^{33 (link),34 (link)}. To decrease calcium influx, calcium-free culture media was used, in which DMEM was substituted with calcium-free DMEM (Life Technologies) with 1mM EDTA. Cadmium (Sigma-Aldrich), a non-selective voltage-gated calcium channel blocker, was added into the medium to a final concentration of 10 μM. Nifedipine (Sigma-Aldrich), an L-type Ca²⁺ channel blocker, was added to a final concentration of 1 μM. To disturb calcium release from internal stores, application of 100 μM Ryanodine (Sigma-Aldrich) or 2 μM Xestospongin C (Sigma-Aldrich) was used to block Ryanodine-sensitive channels or inhibit inositol-1,4,5-trisphosphate (IP3) receptors, respectively.

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)