Cd41 clone ebiomwreg30

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.

Isolation of BFU-E and CFU-E enriched populations was performed as previously described (Zhang et al., 2013 (link), Flygare et al., 2011 (link)). In detail, fetal liver was dissected from embryonic day 14.5 mice and disrupted and homogenized with manual pipetting, followed by lysis of mature red blood cells with ammonium chloride solution (Stem Cell Technologies). Remaining fetal liver cells were stained with biotinylated antibodies against the following surface proteins: CD3e, CD45R, Ly-6G, CD11b, Ter119 (all 5 from the BD-Pharmingen biotin mouse lineage depletion panel), Ly-6A/E (i.e. Sca-1, clone D7, E-Biosciences), CD16/32 (clone 93, E-Biosciences), CD41 (clone EbioMWReg30, E-Biosciences), and Ter119 (E-biosciences). Cells were then incubated with streptavidin-labeled magnetic beads (BD) to deplete cells labeled with biotinylated antibodies. Remaining unlabeled cells were then purified by FACS (staining procedure described below) on a FACS Aria sorter (BD).