Live dead fixable blue dead cell stain kit

Prior to staining-stimulated lymphocytes for flow cytometry analysis, restimulated lymphocytes were treated with 5 μg/mL brefeldin A (Biovision, San Francisco, CA, USA) for 4 h to block cytokine release. Splenic and LN lymphocytes were subjected to a viability stain using a LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (ThermoFisher). Cells were then washed with Dulbecco’s PBS (Gibco, ThermoFisher) plus 10% fetal bovine serum (Atlanta Biologicals) and labeled with mAbs specific for TCR-β, CD4, CD8α, CD19, CD25, TGF-β (BioLegend, San Diego, CA), and CD39 (eBioscience, San Diego, CA). Cells were then fixed and permeabilized using the True-Nuclear Transcription Factor Buffer Set (BioLegend) and labeled with mAbs specific for IFN-γ, IL-17 (BD Pharmingen, San Jose, CA), GM-CSF (eBioscience), IL-10, and Foxp3 (Invitrogen). Fluorescence was acquired on a Fortessa flow cytometer (Becton Dickinson Franklin Lakes, NJ), using FACSDiva software (Becton Dickinson). All samples were analyzed using FlowJo software (BD Biosciences, Ashland, OR).

Expression of PDL-1 (R&D Systems), CD86, CD80 (eBioscence) and I-A [2 (link)] (MHC/H2 class II histocompatibility molecules; BD Bioscience) were analyzed using specific anti-mouse antibodies. Non-specific fluorescence was measured using specific isotype monoclonal antibodies and GBM cells as negative control. Pericytes death was determined using (LIVE/DEAD Fixable Blue Dead Cell Stain Kit, ThermoFisher). Stained cells were analyzed by flow cytometry using a FACS Canto II Flow cytometer (BD Bioscience) and data were analyzed with Kaluza analysis software (Beckman Coulter, Fullerton, CA).

Mandibular dLNs of CD11c-YFP mice were excised, and single cells were obtained by mechanically passing the samples through a 40 μm strainer (Thermo Fisher Scientific, Waltham, MA, United States). Single-cell suspensions were then blocked in FACS buffer containing 1% anti-CD16/CD32 Fc block (Bio X Cell) and were stained with a viability marker (LIVE/DEAD Fixable Blue Dead Cell Stain kit, Thermo Fisher Scientific) for 30 min at RT. Next, samples were stained with fluorophore-conjugated antibodies against CD45, CD11c, dendritic cell inhibitory receptor 2 (DCIR2), CD68, CD3, or respective isotype controls (all BioLegend, or BD Biosciences, San Jose, CA, United States) for 45 min at RT to yield fluorescence minus one stainings. After washing, samples underwent flow cytometric acquisition using a BD LSR II flow cytometer (BD Biosciences). Postacquisition data analysis was performed using FlowJo v9 software (FlowJo LLC, Ashland, OR, United States).

After homogenizing spleens, cells were treated with erythrocyte lysis buffer, meshed through a 35-μm cell strainer, washed with PBS, and analyzed by FACS.
DCs were phenotyped by using fluorochrome-labeled mAbs directed against CD11c (N418; BioLegend), CD80 (16-10A1; BD Pharmingen, Heidelberg, Germany), CD86 (PO3; Thermo Fisher Scientific), PD-1 (J43, Thermo Fisher Scientific), CTLA-4 (UC10-4B9; BioLegend), CD83 (Michel-19; BioLegend), CD40 (3/23; BioLegend), MHCII (2G9; BD Pharmingen) and PD-L1 (MIH6; AbD Serotec, Puchheim, Germany). Dead cells were excluded using the LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific). Following mAb labeling for 30 min at 4 °C and final washing, cells were analyzed on an LSR II flow cytometer.
To measure intracellular cytokines, cells were stimulated with PMA and ionomycin (1 μg/ml each; Sigma-Aldrich, Taufkirchen, Germany) for 4 h in the presence of 3 μg/ml Brefeldin A (Thermo Fisher Scientific), labeled for CD11c, subjected to fixation and permeabilization and subsequently stained with mAbs against IL-10 (JES5-16E3; BD Pharmingen) and IL-12 (C15.6; BD Pharmingen). IFN-γ in T cells was measured by using fluorochrome-labeled anti-IFN-γ (XMG-1.2, BioLegend) and counterstaining with anti-CD4 (RM4-5, eBioscience, Frankfurt, Germany).

Liver tissue was collected and incubated in DMEM (Thermo Fisher Scientific; catalogue number 11965118), 10% FBS (GE Healthcare Life Sciences, Melbourne, VIC, Australia; catalogue number SH30084.03) and 1 mg/mL collagenase (Sigma-Aldrich; catalogue number C5138) for 1 h [32 (link)]. Afterward, leucocyte suspensions were obtained, and immunostaining was performed as described [22 (link),33 (link)]. Cells were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit (Thermo Fisher Scientific; catalogue number L34962, 1/200 dilution). Antibodies are listed in Table 2. Cells were analysed on a custom 10-laser LSR II (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data were analysed with FlowJo software version 9.9 (TreeStar, Ashland, OR, USA) [33 (link)].

0.5-2x10⁶ PBMCs were stained for 15 min at 4°C with different combinations of antibodies (Table S2) and washed before acquisition. 4,6-Diamidine-2-Phenylindole (DAPI) (Molecular Probes, Eugene, USA) or LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, USA) was used to identify dead cells according to the manufacturer’s protocol. Cells were acquired on a FACS Canto II or LSR Fortessa X-20 flow cytometer (BD Biosciences, Heidelberg, Germany) (Figure S1A) (19 (link)). For quality control, CS&T Beads (BD Biosciences), SHPERO Calibration Particles (BD Biosciences) and in some experiments, application settings have been used to obtain reproducible median fluorescence intensities (MFIs). The isotype control of BTLA was measured in a fluorescence minus one (FMO) control approach.