Anti cd45 v450

Individual CD34⁺ CD7⁻ ETPs depleted of CD123^hi cells were deposited by cell sorting (FACSAria Fusion; BD Biosciences) directly into gelatin coated (0.1%; Sigma-Aldrich) 384-well plates (Cell Carrier; PerkinElmer) containing confluent monolayers of either OP9–JAG1 or OP9–DLL1 cells previously treated with mitomycin-C (10 mg/ml; Sigma-Aldrich) for 2 h at 37°C. Cells were kept in culture in RPMI plus 10% FBS supplemented with 100 IU/ml rhFLT3L and 200 IU/ml rhIL-7 for 9 d (OP9–JAG1) or 14 d (OP9–DLL1) and were then stained with anti-CD5-Dylight650 (Novus Biologicals), anti-CD13 biotin (ExBio), anti-CD123 biotin, and anti-CD45 V450 (BD Biosciences). Anti-CD13– and anti–CD123-biotin–coupled antibodies were simultaneously detected after labeling with Streptavidin-Alexa Fluor 546 (Thermo Fisher Scientific). Cells were imaged with an Opera (PerkinElmer) confocal microscope, and images of clonal cultures that contained at least 10 human CD45⁺ cells were then analyzed using ImageJ software. The quantification was done for a maximum of 100 cells/well. The lineage output potential of positive clones was defined by scoring when >70% of CD45⁺ cells were exclusively positive for CD5 (T-lineage) or were positive for CD13 and/or CD123 and either expressed or lacked CD5 (myeloid/DC lineage) or were negative for both markers (undifferentiated).