Cd235a fitc

10⁵ differentiating cells were harvested in phosphate‐buffered saline (PBS) containing 1% bovine serum albumin (BSA) (PBS/BSA) and centrifuged at 200 g for 5 minutes. Cell pellets were resuspended and mixed with the appropriate volume of antibody, CD34‐PE (12‐0349‐41, eBioscience, eBioscience Ltd., Hatfield, UK, http://www.ebioscience.com/), CD43‐APC (17‐0439‐42, eBioscience), CD235a‐FITC (11‐9987‐80, eBioscience), and CD71‐APC (17‐0719‐42, eBioscience), to a final volume of 100 μl PBS/BSA, incubated for 30 minutes then analyzed on a LSR Fortessa (BD Biosciences, Oxford, UK, http://www.bdbiosciences.com/) using FACS Diva. The proportion of enucleated cells present in the culture was assessed using CD235a‐FITC, CD71‐APC antibodies, LIVE/DEAD Fixable Near‐IR Stain (L10119, Thermo Fisher Scientific) and Hoechst dye (NucBlue, Thermo Fisher Scientific). Live CD235a⁺ cells were first gated, then anti‐CD71 and Hoeschst were used to define erythroblasts (CD71⁺/Hoechst⁺), nucleated RBCs (CD71⁻/Hoechst⁺) and enucleated RBCs (CD71⁻/Hoechst⁻) (Supporting Information Fig. S7).

Single cell suspensions were prepared using StemPro Accutase Cell Dissociation Reagent (Gibco) and re-suspended in PBS with 1%BSA and 5 mM EDTA. Cells were blocked with MACS FcR Blocking Reagent (#130-059-901) for 40 min on ice according to manufacturer instructions. In total, 1 × 10⁵ cells were washed and stained with appropriate antibodies (Supplementary Table 1) for 20 min at room temperature. Dead cells were gated out using DAPI. To assess enucleation, single cell suspensions were stained with Hoechst33342 1:20 (Thermo Fisher #R37605) for 20 min, washed with PBS 1%BSA and 5 mM EDTA then stained with CD71-APC 1:200 (Thermo Fisher, 17-0719-42), CD235a-FITC 1:1000 (EBioscience #11-9987) and LIVE/DEAD™ Fixable Near-IR Dead Cell Stain 1:100 (Thermo Fisher #L10119) for 20 min at room temperature. Cells were washed with PBS with 1%BSA and 5 mM EDTA and kept on ice prior to analysis using LSR Fortessa Analyser (BD) and FlowJo Software.

In addition to anti-CD235a, a second probe used was a marker for phosphatidylserine, Annexin V Alexa Fluor 647(ThermoFisher Sci). Following shear stress, cells were suspended to 10⁶ cells in 0.5 mL Ringers solution. The cellular suspension was then labeled with 5 µL of CD235a-FITC(eBioscience) and 5 µL Annexin V Alexa Fluor® 647 according to company protocol. After a 60 min. incubation period at 37 °C, cell and microparticle samples in media were then analyzed by flow cytometry.