Live dead cell fluorescent reactive dye

Anti-mouse monoclonal antibodies (mAbs) to CD16/32 (clone 2.4G2, no. 553142, BD Pharmingen), CD45.2 (clone 104, no. 103131, Biolegend), CD8 (clone SK1, no. 100734, Biolegend), CD11c (clone HL3, no. 557798, BD), CD11b (clone M1/70, no. 101224, Biolegend), CD103 (clone M290, no. 121405, Biolegend), MHCII (clone M5/114.15.2, no. 107628, Biolegend), CD4 (clone GK1.5, no. 553729, BD Pharmingen), CD172a (clone P84, no. 144013, Biolegend), CD45.1 (no. 562452, BD Pharmingen), IRF4 (clone 3E4, no. 11-9858, eBioscience), IRF8 (clone V3GYWCH, no. 17-9852, eBioscience) and rat IgG1 isotype (clone RTK2071, no. 400407, 400426, 400120, Biolegend) were used. For staining of myeloid cells, cells were incubated with mAbs to CD16/32 and live/dead cell fluorescent reactive dye (Invitrogen Molecular Probes, USA) for 20 min at 4_C to block unspecific staining and exclude dead cells. All mAbs were incubated for 30 min at 4_C at predetermined optimal concentrations. For intracellular staining of IRF4 and IRF8, cells were fixed and permeabilized using the FoxP3 intracellular staining kit according to manufacturer’s instructions (eBioscience). Cells were acquired on a BD LSR Fortessa X20 flow cytometer and analysed using Kaluza software (Beckman Coulter).