Fixable viability dye efluor 780

For H2-K^b Ova_257-264 (SIINFEKL)-specific CD8⁺ T-cell analysis, spleen cells were obtained 12 days after tumor inoculation and stained with AF647-coupled dextramer loaded with Ova_257-264 peptide (Dex^OT–I; Immudex, Copenhagen, Denmark), FITC-anti-CD11b, PerCP-anti-CD43, BV421-anti-CD8 (all from BioLegend, San Diego, CA, USA), Fixable Viability Dye eFluor 780, PE-Cy7-anti-CD62L (eBioscience), and BV510-anti-CD44 (Becton-Dickinson).
The frequency of CD8⁺ T cells specific for the H2-D^b-restricted Leader-Gag-derived epitope GagL_85–93 [CCLCLTVFL (33 (link))] was analyzed 14 days after DNA-based immunization in peripheral blood cells after erythrocyte lysis or in spleen cells after tumor cell inoculation. Cells were stained with PE-coupled MHC I tetramer [TetI^GagL; carrying the peptide AbuAbuLAbuLTVFL, in which cysteine residues of the original GagL_85-93 amino acid sequence were replaced by aminobutyric acid (Abu) to prevent disulfide bonding; MBL, Woburn, MA, USA], PerCP-anti-CD43, BV421-anti-CD8, BV510-anti-CD44, PE-Cy7-anti-CD62L (all from BioLegend), and Fixable Viability Dye eFluor 780 (eBioscience).
Data were acquired on a BD FACSymphony A5 flow cytometer (Becton-Dickinson) and analyzed using FlowJo software (TreeStar). Exemplary plots showing the gating strategy are shown in Supplementary Figure 2.

Splenocytes from MLDSTZ-induced diabetic male CD-1 mice were cultured at 37 °C for 5 h with/without LPS (Sigma-Aldrich, 10 ng/mL) and with/without cell activation cocktail (#423304, BioLegend, containing PMA, ionomycin and Brefeldin A, 2 µL per mL cell suspension).
Sorted macrophages were cultured at 37 °C for 24 h with LPS (10 ng/mL) and with or without IL-35 (recombinant IL-35, PeproTech, Cranbury, NJ, USA, 10 ng/mL). cell activation cocktail (2 µL per mL cell suspension) was added 5 h prior to cell collection. The cells were then stained for Arg-1, CD11b, F4/80, MHC-II, and Fixable Viability Dye eFluor^TM 780 (Thermofisher) and acquired on BD LSR Fortessa flow cytometry.

Single cells from thymic glands, PDLNs and spleen were stained with the following surface antibodies: CD11b (M1/70, BioLegend, San Diego, CA, USA), F4/80 (BM8, BioLegend), MHC-II (M5/114.15.2, BioLegend), CD11c (N418, BioLegend), B220 (RA3-6B2, BioLegend) and PDCA-1 (927, BioLegend). The cells were then permeabilized and fixed overnight at 4 °C with Fixation and Permeabilization Buffer (eBioscience, San Diego, CA, USA). The next morning, single cells were stained with antibodies to Arginase 1 (A1exF5, ThermoFisher, Auburn, AL, USA), TNF-α (MP6-XT22, BioLegend), Ebi3 (355022, R&D Systems, Minneapolis, MN, USA) and IL-12p35 (27537, R&D Systems). Fc block (#553142 BD BioSciences, San Jose, CA, USA) was used for both surface and intracellular staining. Fixable Viability Dye eFluor^TM 780 (Thermofisher) was used for detecting live cells. All the samples were analyzed using a BD LSR Fortessa at the BioVis Platform (Uppsala University, Uppsala, Sweden). Data from flow cytometry were analyzed using the Flowlogic version 8.6 software (Inivai Technologies, Mentone, Australia) and FlowJo (Ashland, OR, USA). Gating strategies used for analysis are shown in Supplementary Figures S2 and S3.

The cells were disaggregated and resuspended in 1× buffer, with the concentration adjusted to 1 × 10⁶ cells/mL. Fixable Viability Dye eFluor^TM 780 (Thermo, Cat#65-0865-14), Anti-CD44 (Biolegend, Cat#338807), Anti-CD24 (Biolegend, Cat#311103) were used protected from light. ALDEFLUOR kit (STEMCELL Technologies) was used according to the manufacturer’s instructions to monitor ALDH1 activity. Samples were detected using CYTEFLEX-2 (Backman).

Single-cell suspensions were passed through a 40-µm cell strainer and preincubated with antimouse CD16/CD32 Fc block (1:100, Thermo Fisher Scientific) for 15 min in flow buffer (PBS supplemented with 5% (v/v) FCS) and, subsequently, with the appropriate antibody mix (Supplementary Materials Table) for 20 min on ice.
Dead cells were excluded by staining with either FxCycle^TM Violet Stain (1:1,000, Thermo Fisher Scientific) or Fixable Viability Dye eFluor^TM 780 (1:1,000, Thermo Fisher Scientific) according to the manufacturer’s instructions. All samples were gated on viable cells followed by exclusion of cell doublets and CD45⁺, LYVE1⁺, PDPN⁺ and TER119⁺ cells using BD FACS Diva Software (BD Biosciences). For flow cytometry, samples were recorded on a BD LSR Fortessa or BD FACSCanto II cell analyzer (both BD Biosciences) and flow data were analyzed with FlowJo software (BD Biosciences, v.10). Tumor cell frequencies were calculated either as a percentage of sample-matched lung endothelial cells (TC number), as total TC counts per whole lung or normalized to milligrams of lung tissue using CountBright^TM Absolute Counting Beads according to the manufacturer’s protocol. Cells were sorted using a BD Biosciences Aria cell sorting platform with 100-µm nozzle.