Dmem f 12 glutamax

E12.5 embryos were dissected from pregnant Rosa26^mTmG/mTmG or Rosa26^nTnG/nTnG females mated with Pax3^Cre/+ males. Embryos and PPFs were dissected as previously described (Bogenschutz et al., 2020 (link)). Briefly, embryos were dissected from yolk sacs in DMEM/F-12 GlutaMAX (Invitrogen) pre-warmed to 37°C. To isolate the trunk region, embryos were cut posterior to the forelimbs and just anterior to the hindlimbs, leaving liver largely attached to the trunk. Heart and lungs were removed from the thoracic cavity with forceps and the trunk trimmed to expose the PPFs sitting cranial to the liver. Trunks were pinned to a 6 mm dish coated in Sylgard and PPF pairs manually isolated with forceps from the body wall, septum transversum, and underlying liver. Each PPF explant pair was then placed in a single well from a 96-well plate with 100 μl of media. Growth of both PPF fibroblasts and myogenic cells was promoted using DMEM/F-12 GlutaMAX (Invitrogen), 10% FBS, 50 μg/ml gentamicin, and 0.5 nM FGF. PPFs were grown in a 37°C incubator overnight and then imaged on an ImageXPress Pico automated cell imager (Molecular Devices) or a Leica SP8 confocal microscope, for 1–4 days, changing media in the wells every 2 days.

ADSCs used for the experiments were previously isolated using methods adapted from Bunnell et al. [75 (link)] and Santos et al. [76 (link)] under University of Technology Sydney Human Research Ethics approval Santos-2013000437 (Approval on 02/07/2013). All donor participants volunteered through informed consent for waste lipoaspirate donation as per ethics guidelines and were de-identified for research purposes. Cells were cryo-stored in 90% fetal bovine serum (FBS)/10% DMSO and subsequently revived in 90% Dulbecco’s Modified Eagle’s Medium with Ham’s F-12 and GlutaMAX (DMEM/F12 GlutaMAX, Gibco, Life Technologies, Carlsbad, CA, USA) with 10% FBS (Gibco). Cells which were passaged 6–9 times were grown in 80% DMEM/F12 + GlutaMAX and 20% FBS (Gibco, Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO₂ until at least 80% confluent, then were dissociated from the surface with TrypLE Express (Gibco, Life Technologies, Carlsbad, CA, USA) and re-seeded at 20% confluency on 6-well plates to grow to at least 80% confluency. Each experimental condition was made in triplicate.