Tcrβ pe

Total white blood cells from hematopoietic organs were stained in PBS supplemented with 2% FBS with fluorochrome-conjugated mouse antibodies against specific hematopoietic lineage markers. For the analysis of transplant-receiving mice, WBM was stained with fluorochrome-conjugated mouse antibodies to discriminate cells derived from competitors and donors (CD45.1⁺ and CD45.2⁺ respectively) and GFP expression was used to precisely define donor-derived cells (GFP⁺ CD45.2⁺). Fluorochrome-conjugated mouse antibodies were obtained from Becton Dickinson (Streptavidin/PeCy5, CD117/PeCy7, CD45.1/PE, CD4/PE or PB, CD8/PeCy7 or APC, CD19/APC, B220/APCCy7, TCR_β/PE, CD279/BV421, CD11b/PerCP-Cy5.5, Annexin V/APC, Ki67/PE) and eBiosciences (CD117/APCCy7, Sca-1/APC, CD45.2/APC, Gr1/PE). Hoescht 33342 was obtained from Invitrogen. APC BrdU Flow kit (Becton Dickinson) was used according to the manufacturer’s instruction. Cell sorting was performed either on a MoFlow (Beckman Coulter) or Influx (Becton Dickinson) cell sorter and analysis on a Canto II (Becton Dickinson). FACS data were analyzed by FlowJo Software (v8.8.7).

Single cell suspensions were derived from hepatic homogenate and stained with directly conjugated monoclonal antibodies or isotype controls. Staining for cytokine expression was completed after 4 hours of stimulation with 50 ng/mL Phorbol 12-Myristate 13-acetate (PMA; Promega), 1 μg/mL Ionomycin (Calbiochem) and 10μg/mL Brefeldin A (Gold Bio). Data collection and analysis were done as previously described^{11 (link),63 (link)}. Briefly, cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-AF700 (104), CD11b-PE (M1/70), F4/80-APCef780 (BM8), Ly6C-Percp (HK1.4), Gr1-FITC (RB6-8C5), NK1.1-PECy7 (PK136), TCRβ-PE (H57-597), CD4-APCef780 (GK1.5), CD8-ef450 (53-6.7), TNF-BV650 (MP6-XT22), IL-17A-Percp (17B7) and IL-17F-PE (18F10) [all antibodies from e-Bioscience]. Flow cytometry data were then collected using a LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7).

Samples were stained with a 2.4G2 blocking antibody and monoclonal antibodies in 1% PBS-serum for 20 minutes on ice in the dark. For samples stained with CD1 tetramer, cells were incubated for 15 minutes at room temperature in the dark, followed by 15 minutes on ice in the dark. Samples requiring intracellular staining were then fixed and permeabilized using CytoFix/CytoPerm (BD Biosciences) and stained with intracellular antibodies for 15 minutes on ice in the dark. Events were collected on either a FACSAria (BD Biosciences) or a MACSQuant (Miltenyi Biotec). Data were analyzed using FlowJo (Tree Star Inc.). IL-17A-AlexaFluor488, CD45.2-FITC, HSA-FITC, TCRβ-FITC, TCRVβ2-FITC, CD45.1-PE, PLZF-PE, TCRβ-PE, Foxp3-PE-Cy5.5, NK1.1-PerCPCy5.5, TCRβ-PErCPCy5.5, RORγt-PerCPeFluor710, TCRβ8.1/2-PerCPeFluor710, Ly49G2-PerCPeFluor710, IFN-γ-PE-Cy7, NK1.1-PE-Cy7, T-Bet-PE-Cy7, CD45.1-APC, CD25-APC, IL-4-APC, CD4-APCeFluor780, CD44-APCeFluor780, CD45.2-APCeFluor780, CD90.1-APCeFluor780, CD8α-eFluor450, CD3-eFluor450, Ki67-eFluor450, and TNF-α-eFluor540 were purchased from eBioscience (San Diego, CA). Ly49C/I-FITC, Ly49A/D-PE, and CD4-PerCP were purchased from BD Pharmingen. TCRVβ7-PE was purchased from Biolegend. CD1d tetramer loaded with α-GalCer, CD1d tetramer loaded with PBS-57 as well as unloaded controls were provided by the NIH Tetramer Facility.