Sarcoma derived ecm

Muscle stem cells were isolated by FACS using a triple-negative CD31⁻/CD45⁻/Sca1⁻ and double-positive VCAM⁺/α7-integrin⁺ strategy as described previously (Chakkalakal et al., 2012 (link)). Antibody clones, sources and catalog numbers are described in supplementary Materials and Methods. Cells were seeded in sarcoma-derived extracellular matrix (ECM)-coated 96-well plates in rich growth medium [F10 (Gibco), 20% fetal bovine serum (FBS) (Gibco), (5 ng/ml) FGF2 (R&D Systems)] for behavior analysis. For single cell sequencing experiments, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media [Dulbecco's modified eagle media (DMEM), 10% horse serum] for 18 h prior to library preparation.
Cells for behavior analysis were seeded at 850 cells/well on sarcoma-derived ECM (Sigma-Aldrich) in 96-well plates. Cells were maintained in rich growth media [F10 (Gibco), 20% FBS (Gibco), (5 ng/ml) FGF2 (R&D Systems)]. For single cell sequencing experiments in which cells were activated, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media (DMEM, 10% horse serum) for 18 h prior to library preparation.

All images were performed in a temperature and CO2 controlled unit at 37°C and 5% CO₂. All experiments were performed with a 20X air objective, capturing images with a Hamamatsu C11440-22CU camera (pixel size 6.5 x 6.5 μm) using a Nikon Ti with automated XY stage. MuSCs were seeded at 500-1000 cells/well in 20 wells of an optically clear tissue culture treated, plastic bottomed 96 well plate coated with sarcoma-derived ECM (Sigma) immediately after FACS sorting. After 23 hours to allow for adaptation to culture, media was exchanged for the relevant experimental medium and MuSCs were incubated for 1 hour to adapt. MuSCs were imaged at 20X in DIC for 10 hours with a temporal resolution of 6.5 minutes / frame. Myoblasts were passaged and plated 24 hours prior to imaging at 300-500 cells/well. Media exchanges were performed 1 hr prior to imaging, in the same manner as MuSCs. Myoblast imaging was performed in the same manner as MuSCs. MEFs were similarly plated 16-20 hours prior to imaging at the same density and imaged in the same manner. For FGF2 perturbation experiments, 10 wells of an optically clear plastic bottomed 96 well plate were imaged in growth media with [5 ng/mL] FGF2, and the remaining 10 were imaged in growth media with [0 ng/mL] FGF2. Additional details are available in the Supplemental Methods.