Cd45 af488

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.

Pancreatic tumors were minced and dissociated into single cells by sequentially incubating them in 1) dissociation media for 1h at 37C with gentle agitation, 2) Trypsin 0.25% for 10 min at 37°C, and 3) ACK lysis buffer (ThermoFisher) for 2 min at room temperature to remove erythrocytes. Dissociation media was prepared freshly as follows: DMEM supplemented with 1 mg/mL Trypsin inhibitor (Gibco), 1 mg/mL Dispase II (Gibco), 1 mg/mL Collagenase IV (Gibco) and 0.025 mg/mL DNAse (Sigma). Final suspensions were passed through a 100 μm nylon mesh to remove aggregates. For labeling, cells were incubated on ice with Mouse Fc Block (BD Biosciences, 1/200) followed by antigen-specific antibodies, in FACS buffer (PBS with 1mM EDTA and 0.5% BSA). Cells were sorted at the Salk Institute FACS core facility on a BD Influx cell sorter. DAPI (Molecular Probes) was used to exclude dead cells. For labeling, the following antibodies were used: CD45-AF488, EPCAM-AF647, and PDGFRα-PE (Biolegend, 1/200).

The activated/rested cells were plated into a 96-well round bottom plate and pelleted at low speed. Cells were resuspended in chilled media containing titrated HS-131 or HS-198 (in 1% DMSO) and stained for 30 min at RT. Fluorochrome tagged inhibitors and media were washed away with a PBS wash, followed by a PBS wash containing 3% NMS. Surface antibody stains were performed in 3% NMS/PBS for 30 min followed by PBS washes (2×) (Table 2). Samples were analyzed on BD FACSCanto II flow cytometer. Permeabilized samples were prepared as previously described followed by fixation in 4% Formaldehyde/PBS overnight at 4 °C. Minimum gating of 2X10⁵ cells/sample. Sample analysis was performed with Flowing Software and FlowJo.Table 2

Flow cytometry antibodies.

Antibody	Target and fluor	Manufacturer	Concentration
300434	CD3- BV421	BioLegend	1:200
302616	CD25-AF488	BioLegend	1:200
423113	Viability-BV421	BioLegend	1:1000
423102	Viability-BV510	BioLegend	1:1000
310903	CD69-FITC	BioLegend	1:200
302605	CD25-PE	BioLegend	1:200
561105	Ly6G-AF488	BD Biosciences	1:500
103139	CD45-BV605	BioLegend	1:800
563415	IA/IE-BV650	BD Biosciences	1:1500
103121	CD45-AF488	BioLegend	1:1000
300454	CD3-AF488	BioLegend	1:1000
301817	CD14-AF488	BioLegend	1:1000
400625	Rat IgG2b Isotype Control	BioLegend	1:1000