C kit apc

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.

Single cell suspensions of thymus, spleen, and bone marrow (BM) were harvested and counted at various time points. Cells from the spleen, and BM were subjected to ACK lysis to remove red blood cells. All flow cytometry specimens were incubated with anti-Fcγ III/II receptor (2.4G2) blocking antibody prior to staining. Samples were labeled using combinations of the following antibodies: CD4, CD8, CD3, CD44, CD25, B220, AA4.1, CD45.2, CD45.1, CD45, Sca-1, c-kit (APC (eBioscience) or PE), and IL7Rα (eBioscience), FLT3 (eBioscience), CCR9 (R&D systems), CD11c, CD31, Gr-1, Ki-67, Bcl-2 (Biolegend), CXCR4 (BD or Biolegend), CD150 (eBioscience), CD47 (Biolegend). For DN and ETP/LSK cells subset determination, mature cells were labeled with biotinylated lineage markers: TER, CD8α, H57, Gr1, Mac1, NK1.1, IgM, CD19, B220, CD3, and CD11c. Biotinylated antibodies were developed with streptavidan pacific blue (Invitrogen). All antibodies were purchased from BD unless indicated otherwise. Isotype controls were utilized for all rare populations, including florescence minus one controls. Four, five, and six color panels were acquired on a LSR II flow cytometer (BD Biosciences). All data was analyzed using FlowJo software (Treestar Software).

HA-tagged Etv2 overexpression embryonic stem cell (ESC) lines were generated in our laboratory and have been described previously (Koyano-Nakagawa et al., 2012 (link)). Murine Gata2 coding region was subcloned into the p2Lox vector and then electroporated into A2Lox Cre mES cells to generate the Gata2 overexpressing ES cell line (Iacovino et al., 2011 (link)). The murine Etv2 gene and the murine Gata2 gene were linked through the viral 2A peptide sequence to engineer the Etv2-Gata2 construct, which was utilized to establish the Etv2-Gata2 overexpression ES cell line. ES cell maintenance, embryoid body (EB) differentiation and immunostaining of EBs were performed as described previously (Chan et al., 2013 (link); Rasmussen et al., 2013 (link)). FACS analysis was performed on a BD FACSAria (BD Biosciences). The antibodies used for FACS include: c-Kit-APC (eBiosciences 12-5986), CD31-PE (BD Pharmingen 553373), CD41-PE (BD Pharmingen 558040), Flk1-APC (eBiosciences 17-5821) and Tie2-PE (eBiosciences 12-5987).