Cd86 bv711 it2

For flow cytometry analysis, adherent cells were recovered by trypsinising or gentle scraping and washed in PBS with 2 mM EDTA (PBS/EDTA). Non‐adherent cells were recovered from culture supernatants by centrifugation for 5 min at 300 g and washed once in PBS/EDTA. Cells were stained with fixable Zombie UV Live/Dead dye (BioLegend) for 6 min at room temperature. Excess stain was quenched with FBS‐complemented DMEM. For MDMs, Fc‐blocking was performed with PBS/EDTA+10% human serum for 10 min at 4°C. Cell surface with CD86‐Bv711 (IT2.2, BioLegend) and HLA‐DR‐PerCpCy5.5 or PE‐Cy7 (L243, BioLegend) staining was performed in PBS/EDTA at 4°C for 30min. Unbound antibody was washed off thoroughly, and cells were fixed in 4% PFA prior to intracellular staining. For intracellular detection of SARS‐CoV‐2 nucleoprotein, cells were permeabilised for 15 min with Intracellular Staining Perm Wash Buffer (BioLegend). Cells were then incubated with 1 μg/ml CR3009 SARS‐CoV‐2 cross‐reactive antibody (a kind gift from Dr. Laura McCoy) in permeabilisation buffer for 30 min at room temperature, washed once and incubated with secondary Alexa Fluor 488‐Donkey‐anti‐Human IgG (Jackson Labs). All samples were acquired on a BD Fortessa X20 or LSR II using BD FACSDiva software. Data were analysed using FlowJo v10 (Tree Star).

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.).