Anti il 10 fitc

From mice given different levels of TBI for 12 hours to 3 days, host myeloid cells (CD11b⁺) from spleen were stained with anti-CD11b-PE, and gated for dendritic cells with anti-CD11c-APC and anti-MHCII-PercpCy55, macrophages with anti-F4/80-FOTC, and myeloid derived suppressor cells with anti-Ly6C-PeC7, and anti-Ly6G-v450. Surface stain for co-stimulatory ligands was performed with anti-CD86-PE, anti-CD80-FITC, anti-CD70-APC, anti-ox40L-PerCP, anti-4-1BBL-v450, anti-ICOSL-PE, and anti-H-2D^b (Biolegend). Surface staining of Pmel-1 cells were stained with anti-Vβ13-APC (BD Biosciences) anti-CD62L-FITC, anti-CD44-PerCpCy5.5, anti-IL-17A-PE, anti-IFN-γ-v450, anti-TNF-α-PECy7 and anti-IL-10-FITC (Biolegend) on day 6. For all intracellular staining of cytokines and transcription factors (RORγt-PE and T-bet-FITC; eBiosciences), cultured Pmel cells were restimulated with 1μM human gp100_25-33 peptide using irradiated splenocytes (1:10 Pmel:Irradiated splenocytes) from C57BL/6 mice for 5 hours. Monensin (Biolegend) was added after one hour of stimulation with the peptide. After surface staining, intracellular staining with antibodies was performed according to the manufacturer’s protocol using Fix and Perm buffers (Biolegend). Data were acquired on FACSVerse or Accuri (BD Biosciences). All data was analyzed with FlowJo software (Tree Star).