Anti cd31 pe

Tumors were digested to single cell suspension enzymatically and filtered twice through 70μm filters. Red blood cells were lysed with ACK solution (Gibco), washed twice with Cell Staining Buffer (BioLegend), and then stained with the fluorophore-conjugated antibodies for 30 min at 4 °C. The excess of unbound antibodies was washed out before acquisition in flow cytometry. Cells were analyzed using ID700 Spectral Cell Analyzer (SONY) and sorted using FACS-ARIA III (BD, Pharmingen). The following antibodies were for flow cytometry: anti-CD45 PAC (30-F11), anti-CD8b APC-Cy7 (YTS156.7.7), anti-CD4 PE(H129.19), anti-NK1.1 APC-Cy7 (PK136), anti-CD11b APC (M1/70), anti-Ly6c PE (HK1.4), anti F4/80 (BM8), anti-CXCR6 APC (SA051D1), anti-GranzymeB, anti-CD90 FITC (30-H12), anti-CD73 APC (TY/11.8), anti-PD-1 APC (RMP1-30) all purchased from BioLegend and used at 1:100 dilution. Anti-CD31-PE (eBioscience, 390, 1:100), anti-GLUT1 (Novus, Fgi.72, 1:100), anti-Cxcl16 (Bioss, BS-1441R, 1:50) and anti-Rabbit IgG AF488 (Invitrogen, A21245, 1:100) were used to identify CAF populations. Ghost Dye Red 710 (Tonbo, Cat #13-0871-T100) was used to exclude dead cells from the analysis.

For surface marker analysis, MSCs were harvested and incubated with specific phycoerythrin (PE)-labeled antibodies for 1 hour. Antibodies anti-CD11b–PE, anti-CD45.2–PE, anti-CD31–PE, anti–Ter-119–PE and anti-CD34-PE, rat IgG2b isotype control–PE and rat and mouse IgG2a isotype controls–PE were purchased from eBioscience (eBioscience, San Diego, California, USA), anti–SCA-1–PE was purchased from PharMingen (San Diego, California, USA), and anti–CD34–PE was purchased from Santa Cruz (Santa Cruz, Dallas, Texas, USA). Cells were analyzed using the LSRII flow cytometer (Becton, Dickinson, New Jersey, USA).
For DNA content estimation, cells were fixed with 70% ethanol/phosphate-buffered saline, treated with RNaseA 0.4 mg/ml (Sigma), and stained with propidium iodide 0.1 mg/ml (Sigma). Labeled cells were analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems). Fresh splenocytes were used as control diploid cells.