Fc blocked

To assess the in vivo MSC population in adult mouse prostates or B6CaP tumors by flow cytometry, the tissue of interest was isolated, mechanically dissociated, digested in RPMI + 10% FBS + 1:10 dilution of collagenase/hyaluronidase for one hour at 37°C, triturated in prewarmed PBS + DNAse I, and filtered through a 40μm cell strainer. If necessary, red blood cells were lysed by briefly incubating the cell pellet in ice-cold ACK lysis buffer (Quality Biological). Cells were Fc-blocked (Miltenyi) for 10 minutes in 4°C. Dead cells were discriminated using the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) and samples were stained with the following antibodies from BioLegend: CD45, CD29, CD105, Sca-1, Ly6G, Ly6C, CD11b, CD11c, CCR2, F4/80, MHC II, CD3, CD4, CD8a, FoxP3, CD44, CD62L as well as CD3 from BD Pharmingen. Stained samples were analyzed on an LSR II flow cytometer (BD) and quantified using FlowJo.

PBMCs (1 × 10⁶) were Fc blocked (130-059-901; Miltenyi Biotec), stained, and acquired on a BD LSR II or a BD LSRFortessa (5 (link)). For intracellular cytokine staining, 1 × 10⁶ PBMCs were cultured in the presence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% FBS at 37°C. After overnight incubation, the cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 5 h, with the addition of brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 (325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosciences), PE-IgG₁ κ isotype control (55749; BD Biosciences), CD196/CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-γ-BV421 (563376; BD Biosciences), IL-17-PE (559502; BD Biosciences), γδ TCR-FITC (118105; BioLegend). Data were analyzed using FlowJo (Treestar).