Cd235a bv421

Differentiated cells were treated with 1X TrypLE™ Express for 5 min at 37 °C to obtain a single a cell suspension. Cell pellets were resuspended in FACS buffer (DPBS with FBS 2%) and cells were counted with a hemocytometer. A final amount of 1 × 10⁵ cells resuspended in 100 µl of FACS buffer with a dilution of 1:20 antibody was used for each analysis. The following cell surface antigens were analyzed for this study: KDR-PE (BD, 560494), CD34-APC (BD, 555824), CD43-FITC (Thermo Fisher Scientific, MHCD4301), CD41a-APCH7 (BD, 561422) and CD235a-BV421 (BD, 562938). Cells were washed using the BD FACS Lyse/Wash assistant (BD) and analyzed using BD LSRII flow cytometer (BD). Size and cell complexity were used to identify cell populations in a scatter graph representation. Single cells were discriminated using FSC-A and FSC-H and live cells were gated from single cell population using DAPI nuclear staining. Analysis of data was performed using FlowJo software (Tree Star Inc.). At least 10.000 events were collected for each analysis.

BM- and CB-derived erythroid progenitor cells were differentiated using the three phase EDM. The differentiation state of the cells was determined by staining the cells with fluorescently conjugated antibodies specific to erythroid surface markers: CD36-FITC (BD Biosciences, 20 µL/test), CD71-APC (BD Biosciences, 20 µL/test) and CD235a-BV421 (BD Biosciences, 5 µL/test). The antibody co-stained cells were subjected to FACS (Sony SH800, Sony Biotechnology) and were gated based on differential expression of CD36, CD71, and CD235a on different days of erythroid differentiation: populations 1 and 2 (day 0), populations 3 and 4 (day 4), populations 5, 6, and 7 (day 10 for BM and day 11 for CB). Populations 6 and 7 for BM and CB cells were collected on different days of differentiation to account for slight differences in speed of maturation of BM and CB cells. Purity of the sorted cell populations was assessed by flow cytometry, centrifugation, and cytospin analyses.