Ter119 apc

Cells were suspended in flow buffer (HBSS with 0.1% FBS and DNAse I) for analysis and sorting. Tumor single-cell suspensions were incubated with CD24-PE (553262, BD Biosciences), the endothelial cell marker, CD31-APC (102410, Biolegend), the leukocyte marker, CD45-APC (103112, Biolegend), and the erythrocyte marker, Ter119-APC (116212, Biolegend) according to manufacturer’s instructions for 20 min at 4°C. Following that, cells were rinsed and resuspended in flow buffer before sorting or analysis by FACSAria or FACSCanto instruments (BD Biosciences). The gating strategy for tumor cell sorting was shown in Figure 1—figure supplement 1B-C. For isolation of CD14⁺ cells, CD14-APC/Cy7 (123317, Biolegend) antibody was used and the gating strategy shown in Figure 5C.

Both TA muscles from 1 mouse were pooled, finely minced, and digested for 60 minutes at 37°C in DMEM with 0.2% (~5500 U/mL) collagenase type II and 2.5 U/mL dispase II. The resulting digest solution was filtered through a 70 μm cell strainer and then centrifuged at 350g for 5 minutes. Cells were resuspended and incubated for 30 minutes on ice with primary antibodies, including Sca-1-APC (BioLegend clone D7; 108112), CD45-AF488 (BioLegend clone 30-F11; 103121), CD11b-APC (BioLegend clone M1/70; 101212), Ter119-APC (BioLegend clone TER-119; 116212), CD29/β1-integrin-PE (BioLegend clone HMb-1; 102208), and CD184/CXCR-4-BIOTIN (BD Biosciences lot 6336587; 551968). Cells were then washed, centrifuged, and resuspended in PECy7-STREPTAVIDIN secondary antibody (eBioscience, Thermo Fisher Scientific, lot 4290713; 25-4317-82). The stained cells were filtered through a 35 μm cell strainer, propidium iodide viability dye was added (1 μg) (Thermo Fisher Scientific), and FACS was performed using a BD FACSAria III Cell Sorter (BD Biosciences). MuSCs, classified here as APC/FITC (Sca-1, CD11b, Ter119, CD45) double-negative and PE/PECy7 (β1-integrin/CXCR-4) double-positive cells, were sorted into TRIzol reagent (Thermo Fisher Scientific) and stored at –80°C.