Cd45 fitc

PBMCs (7 × 10⁵ cells) were stained with certain antibodies against L/D-FVS510, CD45-FITC, CD4-PE, CD25-BV421 (all from Biolegend, United States) to analyse the Treg frequency, and FOXP3-APC (EB). The impacted cells were run with BD FACSCalibur and BD FACSAria III flow cytometers (BD Biosciences, United States). Using positive gates and gating controls by Fluorescence minus one (FMO) controls. For Th17 cell analysis, cells were incubated with phorbol-12-myristate-13-acetate (PMA) (50 ng/mL), ionomycin (1 μg/mL), and monesin (1.7 μg/mL; Sigma-Aldrich, United States) at 37°C for 6 h to activate T cells and stimulate the accumulation of intracellular IL-17. Then, the cells were stained with specific antibodies against L/D-FVS510, CD45-FITC, CD4-PE (all from Biolegend, San Diego, California, United States), and IL-17A-APC (EB). The FACSCalibur flow cytometer (BD Biosciences) was used to analyse the stained cells.

TIC and immune cell populations were harvested from the same mice for each experiment. Tumor cell sub-populations were sorted by flow cytometry using CD34-biotin (eBioscience), CD45-FITC, PDGFRα-APC and CD31-PECy7 (Biolegend) antibodies followed by streptavidin-PE. TAMs were isolated using CD45-FITC, CD11b-PECy7 (Biolegend), and F4/80-biotin, followed by streptavidin-PE.

This process was a modification of a well-established protocol^{12 (link), 39 (link)}. Briefly, minced prostate tissues were digested in 1 mg/mL collagenase (Sigma-Aldrich) in RPMI-1640 (Gibco) media containing 10% FBS (Corning) with shaking at 37°C for 2 hours, followed by trypsinization. Dissociated cells were passed through 20G needles and 40 μm cell strainers to eliminate aggregates, followed by removal of red blood cells by ACK buffer. To enrich murine bPSC, isolated cells were stained with Zombie Violet Live/Dead Fixable Viability Dye (Biolegend) in PBS, followed by incubation with 1:100 diluted fluorescence-conjugated specific antibodies (Biolegend): CD45-FITC (#103108), CD31-FITC (#102506), Sca-1-APC (#122512) and CD49f-PE (#313612). Human prostate samples from unidentified patients were acquired from Indiana University School of Medicine Tissue Repository under IRB-approved protocols. Isolated human prostate cells were stained with Zombie UV Fixable Viability Dye (Biolegend), CD45-FITC (#304006), EpCAM-PE (#324206), CD26-APC (#302710) and CD49f-BV421 (#313624) as described in detail by Strand et al.^{40 (link)}. Once stained, fluorescence activated cell sorting was performed on the BD FACSAria under sterile conditions.

The tumors were collected and minced manually into small pieces. A single-cell suspension from tumors were obtained by passing them through the 70 µm and 40 µm cell strainers (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed using a 0.15 M ammonium chloride solution. Dead cells were removed by centrifugation using the Lympholyte-M gradient (Cedarlane, Ontario, Canada). 7-AAD (7-aminoactinomycin D) Viability Staining Solution (BioLegend, San Diego, CA, USA) was used to stain nonviable cells 10 min before running the flow analysis. To identify the subpopulations of T lymphocytes and NK cells, the following antibodies were used: FITC-CD45, PE-Cy7-CD4, APC-CD8, and PE-CD49b (BioLegend). In the flow cytometric analyses (BD FACSCanto, BD, Franklin Lakes, NJ, USA), the gates dividing negative from positive cells were based on isotype antibody control probes [33 (link)].

For multi-parametric flow cytometry, immunofluorescence/immunohistochemistry and DEPArray analyses, primary antibodies were obtained from the following sources: FITC-CD45 (#304054; 1:200), FITC-CD34 (#343504; 1:200), FITC-CD105 (#323204; 1:200), FITC-CD90 (#328108; 1:200), FITC-CD73 (#344016; 1:200), FITC HLA-A/B/C antibody (#311404; 1:200), PerCP/Cy5.5-CD146 (#342014; 1:100), PE-Human NG2/MCSP (#FAB2585P, 1:100), BV421-Ki67 (#350506; 1:100), were obtained from Biolegend, Inc. APC-Cy7-CD44 (#103028, 1:100) BV510-CD24 (#311126, 1:100), PE-Pan-Cytokeratin (#5075, 1:100) were purchased from Cell Signaling Technology, Inc. Anti-Mel-A antibody (# AC12-0297-03; 1:200) was obtained from Abcore, and FITC-Anti-S100 (#ab76749; 1:50) was purchased from Abcam. For immunohistochemistry, anti-human, anti-Mel-A antibody (# ab51061; 1:100), HLA-ABC (#565292; 1:100) were obtained from BD Biosciences, Inc. (San Jose, CA, USA). Anti-mouse secondary IgG anti-human antibodies used for IHC staining were received from Santa Cruz Biotechnology, Inc. Antibodies for immunofluorescence staining were obtained from Cell Signaling Technology, Inc. (1:500 dilution of stock solution), as previously described [3 (link),22 (link)]. NRF2 inhibitor ML385 (#2114) was obtained from Cayman, Inc. while Nodal inhibitor Lefty (746-LF/CF) was purchased from R&D Systems, Inc. (Waltham, MA, USA).

CAR T cells and ChTCR T cells were harvested and washed once with phosphate buffer saline (PBS). Cells were stained with anti-human CD45 antibody (clone HI30), so that each receptor was stained with a unique CD45 fluorescent barcode (single or double stain with APC-CD45 (Biolegend, 304012), PE-CD45 (BD, 555483), PerCp-Cy-5.5-CD45 (BD, 564105), FITC-CD45 (Biolegend, 304006), BUV805-CD45 (BD, 612891)). Cells were washed three times, pooled 10⁷ cells total. Cells were stained with 5μM indo-1AM dye (Invitrogen, I1223) in calcium stain buffer (phenol-free RPMI, 1%FBS, 0.5 mM probenecid (Sigma, P8761–100G), 10 mM HEPES) at 37° for 45 minutes. Cells were then washed twice with calcium stain buffer, resuspended in 4 mL calcium stain buffer, and split into 4 tubes. Prior to calcium measurement, cells were incubated with biotinylated proteins/antibodies (1 μg/mL CD19-biotin (Accro Biosystems, CD9-H82E9) , 0.5 μg/mL anti-CD28-biotin (Biolegend,302904)) for 5 minutes at 37°. Baseline indo-1AM fluorescence was measured for 30 seconds, before addition of 20μg/mL avidin to crosslink biotinylated proteins. Calcium flux was measured for 5 minutes before addition of 1X cell stimulation cocktail (Invitrogen, 00–4970). Multiplexed populations were deconvoluted and calcium plots generated in FlowJo software (BD), and area under the curve measurements made using Prism software (GraphPad).