Zombie aqua fixable dye

Flow cytometry was performed as described and using gating strategies in ref. 32 . Briefly, for analysis of surface markers on iPS-DCs or Monocyte-derived DCs, cells were stained with Zombie Aqua fixable dye (BioLegend), Fc receptors were blocked using human TruStain FcX (BioLegend), and cells were then subsequently stained for iPS-DC surface markers anti-CD11c-FITC (Bu15, Biolegend) and anti-CD141-APC (M80, Biolegend) or Monocyte-derived DCs surface markers anti-HLA-DR (L243, Biolegend) and anti-CD209 (DC-SIGN) (9E9A8, Biolegend). For the detection of TLR2, cells were also stained anti-TLR2-PE (TL2.1, Biolegend) or TLR2-APC (W1514C, Biolegend) prior to fixation with 4% paraformaldehyde. Data were acquired using an Attune NxT flow cytometer (Thermo Fisher). Electronic compensation was performed with antibody (Ab) capture beads (BD Biosciences) stained separately with individual monoclonal antibodies used in the experimental panel. Data were analysed using the FlowJo software (TreeStar, Inc.).

For determination of apoptotic and necrotic cells, an Annexin V/Zombie assay (BioLegend) was used. Heparinized human whole blood samples were incubated with 6-FAM-labeled PFOB-NE at a dilution of 1:20 at 37 °C under constant shaking for 4 h. 200 µL of whole blood was taken, placed on ice and lysed 3 times for 15 min. Afterwards, leukocytes were re-suspended in PBS and incubated with Zombie Aqua Fixable Dye (BioLegend, 1:100) for 15 min. Cells were then washed with FACS-buffer (2% FCS, 2 mmol/L EDTA in PBS), re-suspended in Annexin V binding buffer and stained with APC-labeled Annexin V (BioLegend, 1:20) for 15 min. Flow cytometry was performed as described above. In our gating strategy neutrophils, monocytes or lymphocytes were located in the FSC and SSC, cell duplicates and dead cells were excluded and necrotic, apoptotic and living cells were discriminated by Annexin V and Zombie staining. A representative example of the gating strategy is shown in supplementary Figure S4.

For analysis of surface markers on iPS-DCs, cells were stained with Zombie Aqua fixable dye (BioLegend), Fc receptors were blocked using human TruStain FcX (BioLegend), and cells were then subsequently stained for surface markers with a combination of the following antibodies: anti-human HLA-DR-Alexa Fluor 488 (AF488) (L243; BioLegend) or CD14-FITC (M5E2; BioLegend), CD83-PerCP/Cy5.5 (HB15e; BioLegend) or CD1c-PerCP/Cy5.5 (L161; BioLegend), CD141-PE/Cy7 (M80; BioLegend) or DC-SIGN-PE/Cy7 (9E9A8; BioLegend), or XCR1-PE (FAB8571; Bio-Techne), CD11c-APC/Cy7 (Bu15; BioLegend), CLEC9A-APC (8F9; BioLegend), CD86-BV711 (IT2.2; BioLegend), CD303-BV785 (201A; BioLegend) or HLA-DR-BV785 (L243; BioLegend), and HLA-A,B,C-Pacific Blue (W6/32; BioLegend). For the detection of IRF5 or IAV nucleoprotein, cells were stained with Zombie Aqua fixable dye, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100, followed by incubation with human TruStain FcX and staining with IRF5-AF647 (EPR6094; Abcam) or anti-influenza A virus nucleoprotein antibody (431; Abcam) in 0.1% Triton X-100 solution. All data were acquired using an LSRFortessa flow cytometer (BD Biosciences). Electronic compensation was performed with Ab capture beads stained separately with individual MAbs used in the experimental panel. Data were analyzed using the FlowJo software (TreeStar, Inc.).