Anti cd45 bv785

Blood samples were incubated with ACK lysis solution to remove erythrocytes. Single-cell suspension of splenic cells was obtained by gently pressing spleens through 70 μm cell strainers (Corning, cat. no. 352350). Bone marrow cells were harvested from one femur per mouse. Peritoneal lavage was performed by repeatedly flushing the peritoneal cavity with 10 ml PBS/2% FCS. Single-cell suspensions from the different tissues were resuspended in PBS/2% FCS and briefly incubated with Fc blocking antibodies (clone 2.4G2, purified in-house). Cells were then stained with the following fluorescently labelled monoclonal antibodies: anti-CD8α-FITC (ThermoFisher, cat. no. 11-0081-81), anti-TCRβ-PE (BioLegend, cat. no. 109208), anti-CD11b-PerCP-Cy5.5 (BioLegend, cat. no. 101228), anti-Gr-1-APC (ThermoFisher, cat. no. 17-5931-82), anti-CD19-PE-Cy7 (BioLegend, cat. no. 115520), anti-CD45-BV785 (BioLegend, cat. no. 103149), anti-CD4-BV711 (BioLegend, cat. no. 100447), anti-CD11c-BV605 (BioLegend, cat. no. 117333) and anti-Ly-6G-BV421 (BioLegend, cat. no. 127628). For nonviable cell exclusion, Fixable Viability Dye eFluor^® 780 (ThermoFisher, cat. no. 65-0865-18) was used. Stained cells were then fixed with 4% formalin and resuspended in PBS/2% FCS for flow cytometric analysis using a LSRFortessa (BD Biosciences). FACS data were then analysed with FlowJo software (RRID:SCR_008520, version 10.0.7).

TILs were suspended in AIM-V (Thermo Fisher Scientific, Tokyo, Japan) supplemented with 10% AB serum (Biowest, Nuaillé, France) and then treated with Golgi STOP (1:1000), PMA (50 ng/mL), and ionomycin (1 µM) for 4 h. Harvested cells were stained with Zombie Yellow Fixable Viability kit (BioLegend), anti-CD45-BV785, anti-CD45RA-APC-Cy7 [HI100], anti-PD-1-PE-Cy7, and anti-Tim3-PE. After fixing with Foxp3/transcription factor staining buffer set (Thermo Fisher Scientific), cells were stained with anti-CD3-BV421 [UCTH1], anti-CD4-APC [RPA-T4], CD8-BV711 [RPA-T8], anti-IL-2 [PerCP-eFluor710], anti-TNFα-BV510 [Mab11], and anti-IFNγ-FITC [4 S.B3].

Mouse tumors were minced and incubated in collagenase from Clostridium histolyticum (Sigma), 2 mg/ml, and DNase I from bovine pancreas, 25 μg/ml (Sigma) in HBSS (Sigma) for 20-30 min at 37°C. The lysate was strained through 70 μm strainers (Fisher Scientific). Cells were counted and 0.5-1x10⁶ cells stained in PBS, 2.5% BSA, 2 mM EDTA after blocking with Trustain (Biolegend) for 15 min. Staining antibodies were diluted 1:200 unless differently specified: anti-CD45-BV785 (1:100, Biolegend), anti-CD3 PE-Cy7 1:50 (Biolegend), anti-CD4 BV605 1:100 (Biolegend), anti-CD8 APC (eBioscience), anti-CD11b BV650 (Biolegend), anti-CD11c FITC (eBioscience), anti-F4/80 PE (Biolegend), anti-Ly6C eFluor450 (eBioscience, 1:100), anti-Ly6G(Gr1) AF700 (eBioscience), anti-CD19 PerCP-Cy5.5 (eBioscience), anti-MHCII APC-Cy7 (Biolegend). Viability was assessed with Fixable Viability Dye eFluor506 (eBioscience) diluted 1:500. Staining was performed for 30 min at 4°C. The cells were finally washed, fixed and analyzed on a BD LSR Fortessa cytometer. Appropriate Fluorescence Minus One (FMO) controls were used in these experiments. Analysis was performed with the FlowJo software. Human omental tissue was processed and stained similarly as previously described (Böhm et al., 2016 (link)).