Cd8α efluor450

Flow cytometry was utilized to determine cell phenotype and cell proliferation. Cells were stained for surface markers using fluorochrome conjugated antibodies for 30 minutes on ice, washed, acquired using FACSCantoII (BD Biosciences) and analyzed using FlowJo v10.4.2 (Tree Star LLC). Antibodies and their specific clones used were: CD3-FITC (17A2, eBioscience), CD3-PerCPCy5.5 (145–2C11, Tonbo Biosciences), CD4-Pacific Blue (RM4–5, eBioscience), CD4-PerCPCy5.5 (RM4–5, Tonbo Biosciences), CD8α-APC-Cy7 (53–6.7, eBioscience), CD8α-eFluor450 (53–6.7, eBioscience), CD11b-PercpCy5.5 (M1/70, Tonbo Biosciences), CD11c-APC-eFluor780 (N418, eBioscience), B220-PECy7 (RA3–6B2, eBioscience), CD44-PECy7(IM7, eBioscience), Foxp3-FITC (FJK-16s, eBioscience), I-A^b-APC(AF6–120.1, eBioscience), interferon-γ-APC (XMG1.2, eBioscience), violet proliferation dye (VPD450, BD Biosciences), and aqua live/dead dye (Molecular Probes).

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.