Hanks balanced salt solution (hbss)

Timed-pregnant mice and mouse colonies for timed matings were maintained as above, and all procedures were IACUC-approved. Muscle from the limbs of neonatal mice less than 24 h old was dissected away from other tissues into Hanks' balanced salt solution (HBSS; Corning, #21-023-CV), the HBSS was aspirated, and the muscle was minced with sterile scissors and incubated in freshly prepared 2% Type II collagenase (Worthington Biochemical, #LS004174) in HBSS for 30 min with vortexing every 10 min. Cells were pelleted by centrifugation at 1800 × g for 5 m at room temperature, resuspended in HBSS, and re-pelleted as above. Cells were resuspended in DMEM supplemented to contain 6% FCS, 2 mM L-glutamine (Corning #25-005-Cl), and 10 μg/ml gentamycin, and passed through a 100 μm cell strainer (Falcon, #352360). Cells were resuspended in supplemented DMEM and plated in 8-well poly-D-lysine-coated chamber slides for immunofluorescent microscopy. After incubation at 37°C in 5% CO₂ for two days, media was replaced with DMEM supplemented as above (for undifferentiated skeletal muscle cell cultures) or DMEM supplemented as above but at 3% instead of 6% FCS (for differentiated skeletal muscle cell cultures). Cultures were incubated an additional two days before use to allow differentiation, and were never passaged.

Mesenchymal cells were isolated from the pooled proximal halves of the small intestine from 3–6 wildtype or Pdgfra^H2B-eGFP pups. Timepoints harvested from wildtype tissue include P1, P4, P5, P9, and P14, and from GFP+ isolated Pdgfra^H2B-eGFP tissue include P2, P5, and P14. After manual stripping of external muscles (MP) and serosa, whole adult Myh11^Cre(ER-T2);R26R^L-L-TdTom; Pdgfra^H2B-eGFP SI or colon was processed as described.^{30 (link)} Epithelium was denuded by shaking the tissue for 20 min at 37^oC in pre-warmed HBSS (Life Technologies) containing 10 mM EDTA. The remaining tissue was rinsed with HBSS, minced using a scalpel, and digested with gentle rocking for 1 h at 37^oC in 3 mg/mL collagenase II (Worthington, LS004176) diluted in HBSS containing 5% fetal bovine serum (FBS). Extracted cells were centrifuged at 300 g for 5 min, washed with FACS buffer (PBS containing 0.1% BSA), and leukocytes were depleted in ACK Lysis buffer (Gibco) for 3 min. Washed cells were suspended in FACS buffer and, to deplete epithelial and immune populations, stained with conjugated EPCAM (BioLegends, 118214, 1:100) and CD45 (eBiosciences, 17–0451-82, 1:100) Ab for 20 min at 4°C. Cells were sorted on a FACSAria IIII flow cytometer, with gating against DAPI (BD Pharmingen) to identify live cells. Graphs of isolated cell fractions were generated using FlowJo software v10.

Mice were euthanized according to our IACUC protocol, and then main olfactory epithelia were dissected and quickly immersed in ice-cold HBSS (Worthington). Olfactory epithelia were separated from the bone under a dissection microscope and then were minced into small pieces using forceps. Each epithelium was then washed once with HBSS and resuspended in 2 ml of EBSS containing papain (20 U/mL, Worthington) and DNase I (2000 U/mL, Worthington). After 40 minutes of incubation with gentle agitation at 37°C, the suspension was washed with 5 ml of PBS + 0.02% BSA twice and passed through a 30 μm cell strainer (Miltenyi Biotec).