Cd71 fitc

Adherent cells were washed with Dulbecco's PBS (DPBS) (Thermo Fisher, Waltham, MA, #12563011) and treated with Tryp‐LE Select (Thermo Fisher, Waltham, MA, #12563011) at 37°C for 5 minutes, followed by gentle pipetting to complete cell dissociation. The dissociated cells were then washed with DPBS several times before antibody staining for flow cytometry. For suspension hematopoietic cells, gentle pipetting was used to dislodge any loosely adhered hematopoietic cells from the adherent stromal layer, and the cells were washed multiple times with PBS before staining. Cells were stained in ice‐cold fluorescence‐activated cell sorting buffer (PBS, 1% BSA). Antibodies used; KDR‐PE (BD Pharmingen, San Jose, CA, #560872), CD34‐APC (BD, San Jose, CA, #555824), CD144‐PE (BD, San Jose, CA, #561714), CD45‐PE (BD, San Jose, CA, #561866), CD117‐APC (Thermo Fisher, Waltham, MA, clone 104D2), CD117‐PE (BD, San Jose, CA, #555714), CD235a‐FITC/PE (BD, San Jose, CA, Clone GA‐R2 (HIR2)), CD41a‐APC (BD, San Jose, CA, #559777), CD71‐FITC (BD, San Jose, CA, #555536), CD73‐PE (BD, San Jose, CA, #561014), CXCR4‐Biotin (BD, San Jose, CA, #555973), and Streptavidin‐FITC (BD, San Jose, CA, #554060). Flow cytometry was conducted on a Stratadigm S1000EXI or Beckton Dickinson (BD) dual‐laser FacsCalibur flow cytometer. Analyses were conducted using FlowJo v8.7 (FlowJo, LLC) software.

To further validate FACS sorting we determined the different CD71 intensity in CD71^low, CD71^med and CD71^hi FACS-sorted reticulocytes by IFAs. CD71^low, CD71^med and CD71^hi FACS-sorted reticulocytes were washed with PBS-0.05% BSA and incubated with CD71 FITC (BD) for 30 min at 4 °C; after incubation cells were washed twice with PBS-0.05% BSA and smeared. Images were obtained in an Operetta platform (Perkin Elmer) at 60×.
To visualize enucleation on HSC cultures, HSC-derived cultured cells were washed with PBS-0.05% BSA, re-suspended in PBS-0.05% BSA and then stained with anti-human CD71-FITC and DAPI (4,6 diamidino-2-phenylindole) (Life technologies) for 30 min at 4 °C. Cells were then washed twice with PBS-0.05% BSA, smeared on a glass slide and acquired in an Operetta platform at 60×.