Flk1 apc

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).

Flow cytometric analysis for cell surface markers, were done using antibodies CD31-APC (Catalog#17-0311, eBioScience), CD34-FITC (Catalog#11-0341, eBiosience), CD133-FITC (Catalog#11-1331, eBioscience), Flk1-APC (Catalog#17-5821, eBioscience), VECAD-APC (Catalog#17-1441, eBioscience), CD45-APC(Catalog#103111, Biolegend), ckit APC (Catalog#561074,BD Bioscience), DDR2 (Catalog#SC-7555,Santa Cruz) and Vimentin (Catalog#Ab1620, Millipore). For Vimentin and DDR2, a secondary APC conjugated anti-goat antibody was used (Catalog#SC3860, Santa Cruz). Cultured cardiac fibroblasts were dissociated using accutase (Innovative Cell Technologies, Inc.,) and immunostained in FACS buffer (0.1% BSA PBS ) at 1 × 10⁶/ml for 20–30 minutes at 4°C, followed by washing twice with FACS buffer and subsequently analyzed in Beckman-Coulter (Dako) CyAn ADP. Data obtained was analyzed and represented using Flowjo software.