Cd31 pacific blue

Cells were collected from hindlimb bone tissue as described in “Flow cytometry and single-cell RNA-seq,” resuspended in buffer solution (PBS + 2% FBS + 2 mM EDTA), and blocked with recombinant Fc protein (1:100 dilution, BD Biosciences, catalog no. 553142). Cells were then incubated with the primary antibody solution (1:100 dilution; PDGFRA-APC, Thermo Fisher Scientific, catalog no. 17-1401-81; SCA1-FITC, Thermo Fisher Scientific, 11-5981-82; CD45-BV450, BD Biosciences, catalog no. 560697; TER119-BV450, BD Biosciences, catalog no. 560504; CD31-Pacific Blue, BioLegend [San Diego, CA, USA], 102422; DAPI, Sigma, catalog no. 10236276001) for 30 minutes on ice, washed with buffer solution, and analyzed using the BD FACS-Canto system (BD Biosciences). Specimens were analyzed in trip-licate with at least 100,000 events per replicate.

Cell populations from mammary glands and tumors were assessed using flow cytometry. Tissues and tumors were digested using 500units/ml collagenase type II and IV and 20μg/ml DNase and incubated at 37°C for 90 minutes prior to filtering through a 100μm filter. For the endothelial/epithelial panel, cells were stained with CD45 (BV510; Biolegend #103137), CD31 (Pacific Blue; Biolegend #102422), PDPN (APC; Biolegend #127410), CD49f (PerCP; Biolegend #313617), EpCAM (APC-Cy7; Biolegend #118218), and SEMA7A (FITC; Abcam #26012). For the macrophage panel, cells were stained with CD45 (BV510), CD11b (PerCP; Biolegend #101229), F4/80 (APC-Cy7; Biolegend #123117), CD64 (Pacific Blue; Biolegend #139309), MerTK (FITC; Biolegend #151503), and PDPN (APC). Cells were analyzed on a Beckman Coulter CyAn ADP© using Summit© software in the UCCC Flow Cytometry Core or the Tamburini Laboratory at the CU Anschutz Medical Campus. Data were analyzed using FlowJo analysis software.