Cd45.2 efluor 450

Mouse thymocytes and splenocytes were isolated in RPMI containing 5% fetal calf serum, followed by red blood cell depletion using the ACK lysing buffer (Gibco). Flow cytometric analysis was performed on cells according to a previously described procedure (Jiang et al., 2011 (link)). Antibodies used for analyses: CD3 pacific blue (BD, clone 500A2, 558214), CD8 APC-Cy7 (BD, clone 53-6.7, Cat. No. 557654), CD4 PE (BD, clone GK1.5, Cat. No. 553730), CD80 FITC (BD, Cat. No. 553768), mouse Vβ TCR screen panel (BD, Cat. No. No. 557004), CD25 APC (eBioscience, clone PC61.5, Cat. No. 17-0251-82), Foxp3 FITC (eBioscience, clone FJK16a, Cat. No. 11-5773-82), CD3e FITC (eBioscience, clone 145-2C11, 11-0031-82), CD274 PE (eBioscience, B7-H1, PD-L1, clone MIH5, 12-5982-81), ly-6G (Gr-1) Alexa Fluor 700 (eBioscience, clone RB6-8c5, 56-5931-80), CD11b PE-Cy7 (eBioscience, clone M1/70, 25-0112-81), CD11c 780 (eBioscience, clone N418, 47-0114-80), CD49f (intergrin alpha 6) PE-Cyanine 7 (eBioscience, clone GOH3, Cat. No. 25-0495-80), CD45.1 PE-Cy7 (eBioscience, clone A20, 25-0453-82), CD45.2 eFluor 450 (eBioscience, clone 104, Cat# 48-0454-82), and FITC conjugated UEA1 (Vector, Cat. No. FL-1061). Labeled cells were analyzed on a LSR-II Flow Cytometer (Becton Dickinson). Data were analyzed using FlowJo software.

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.