Cd45.2 apc efluor780

For immunophenotypic analysis of lineage positive cells, PB, BM and spleen samples were processed with 1× red blood cell lysis buffer, and then incubated with CD11b-PE-cy7 (25–0112-81, eBioscience), Gr1-eFluor450 (48–5931-82, eBioscience), CD3-PE (12–0031-83, eBioscience) and B220-APC (17–0452-82, eBioscience). To distinguish donor from recipient hematopoietic cells, PB were stained with CD45.1-Brilliant Violet 510 (110741, BioLegend), and CD45.2-APC-eFluor780 (47–0454-82, eBioscience) or CD45.2-eFluor450 (48–0454-82, eBioscience). For HSPC analysis, BM cells were washed and incubated for 30min with biotin conjugated lineage markers (CD11b, Gr1, Ter119, CD3, B220, mouse hematopoietic lineage biotin panel (88–7774-75 eBioscience)), followed by staining with streptavidin eFluor780 (47–4317-82, eBioscience), Sea-1-PE (12–5981-82, eBioscience), c-Kit-APC (17–117181, eBioscience), Flk2-PE-Cy5 (15–1351-81, eBioscience), CD150-PE-Cy7 (115914, BioLegend) and CD48-FITC (11–0481-85, Affymetrix) or CD48-eFluor450 (48–0481-80, eBioscience). SLAM-HSC were identified based on expression of Lin⁻Sca⁻1⁺c-Kit⁺CD150⁺CD48⁻. MPP2, MPP3 and MPP4 were identified based on expression of Lin⁻Sca^_1⁺c-Kit⁺Flk2⁻CD150⁺CD48⁺, Lin⁻Sca⁻1⁺c-Kit⁺Flk2⁻CD150⁻CD48⁺ and Lin⁻Sca⁻1⁺c-Kit⁺Flk2⁺CD150⁻CD48⁺, respectively.

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.