C kit clone 2b8

Eight-week-old mice were used for FACS analyses of the BM. Single BM cell suspensions were stained using anti-mouse Abs against B220 (clone RA3-6B2; eBioscience), CD19 (clone 1D3; eBioscience), CD43 (clone S7; BD Biosciences, Franklin Lakes, NJ), IgM (clone RMM-1; BioLegend), IgD (clone IA6-2; BioLegend), c-Kit (clone 2B8; eBioscience), Flk2 (clone A2F10; eBioscience) and Ter119 (clone Ter119; eBioscience). The gating was done as described (17 (link), 18 ).

EBs were collected on day 4, 6 and 8 for flow cytometry analysis. For surface marker staining, EBs were trypsinized and incubated with antibodies for 30 min on ice. Propidium iodide (PI) (1 μg/ml, Sigma) was added to differentiate between live and dead cells. Only live cells (PI−) were counted. For intracellular staining, dissociated cells were first fixed with 1% paraformaldehyde for 15 min at room temperature and permeabilized with ice-cold 90% methanol at −20 ºC overnight. Antibody staining was performed on the next day as described above except that PI was not added. Fluorescence activated cell sorting (FACS) were performed using a BD FACSAriaII (BD Biosciences, San Diego, CA) and data were analyzed using FlowJo (Tree Star, Ashland, OR). Antibodies used: Flk-1 (clone Avas12a1, BD Biosciences, San Diego, CA), PDGFRα (clone APA5, BD Biosciences), c-Kit (clone 2B8, eBioscience, San Diego, CA), CD41 (clone eBioMWReg30, eBioscience), and cardiac troponin T (clone CT3, Developmental Studies Hybridoma Bank, Iowa City, IA). The cardiac troponin T antibody was developed under the auspices of the NICHD and maintained by the University of Iowa.