Pdgfrα pe

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).

For staining, cells were washed with FACS buffer. Antibodies were diluted in 50 μL FACS buffer and added to the cells for 30 minutes at 4°C. After incubation, cells were washed twice with FACS buffer to remove unbound Ab. Cells were then resuspended in FACS buffer and analysed using a FACS Canto II (BD Biosciences). All analysis was performed using FlowJo. The monoclonal antibodies used were: gp38‐PE (e‐Biosciences), Syndecan‐2‐APC and NG2‐PE (R&D), CD105‐PE, MHCII‐FITC, CD29‐PeCy5, CD86‐AF488 (Biolegend), PDGFRα‐PE, CD14‐APC, CD34‐PE, CD15‐FITC, MHCI‐PeCy7, Cd73‐PE, CD90‐PE, CD45‐FITC, CD11b‐PeCy7, CD80‐PeCy7 and CD117‐PE (BD Biosciences).