Clone wm53

Approximately 100,000 cells each day per culture replicate were harvested. Media were removed by washing with staining buffer (PBS + 0.25% BSA) and cells were labeled according to product specifications, incubating at 4 °C. Cells from erythroid differentiation were stained daily for CD117 PE at 1:10 dilution (Cat. No.: 340529, Clone 104D2, BD Biosciences), and CD235a at 1:20 dilution (Cat. No.: 559943, Clone GA-R2/HIR2, BD Biosciences). Megakaryocytic differentiation was monitored by staining with CD41 PE at 1:10 dilution (Cat. No.: 555467, Clone HIP8, BD Biosciences), and CD42b APC at 1:10 dilution (Cat. No.: 551061, Clone HIP1, BD Biosciences). Megakaryocytic potential of primary erythroid cultures was determined by measuring CD41a PE at 1:10 dilution (Cat. No.: 555467, Clone HIP8, BD Biosciences) expression in the secondary cultures. Myeloid populations were identified by CD33 expression staining with Alexa Fluor 700 anti-human CD33 at 1:20 dilution (Cat. No.: 561160, Clone WM53, BD Biosciences). For detection of genetic knock-out of CDH1 in HUDEP-2, APC anti-human CD324 at 1:20 dilution (E-Cadherin) (Cat. No.: 324108, Clone 67A4, Biolegend) was used. Samples were acquired on CytoFlex S (Beckman Coulter) and analyzed using FlowJo (Becton Dickinson). All analyses were performed on the live, single events as determined by their profile on the FSC-H, FSC-A, and SSC-A.