Fitc conjugated anti cd71

Antibodies to the following proteins were used: APC-conjugated anti-CD34 (Miltenyl Biotec Cat. 130-090-654; dilution 1:100), FITC-conjugated anti-CD71 (BD Biosciences Cat. 555536; dilution 1:100), PE-conjugated anti-CD235a (BD Biosciences Cat. 340947; dilution 1:500), APC-conjugated anti-CD45 (BioLegend Cat. 304012 Clone HI30; dilution 1:100), FITC-conjugated anti-CD19 (BioLegend Cat. 302206 Clone HIB19; dilution 1:100), and PE-conjugated anti-CD33 (BioLegend Cat. 303404 Clone WM53; dilution 1:100). Dead cells were identified by Hoechst 33258 pentahydrate nucleic acid stain (Invitrogen; dilution 1:10,000) and were excluded.

Prior to the initiation of differentiation protocol, human iPSCs were cultured for at least 15 days on Vitronectin (Invitrogen) coated plates (Corning; TC treated) in STEMPRO hESC media (Invitrogen) supplemented with 20 ng/ml bFGF (RnD). Cultures were maintained in this way for 5–6 passages at high confluency and split 1 in 4 when over 85% confluent. Human iPSCs were differentiated as published by Olivier et al.^{12 (link)} and harvested on day 21 of the differentiation protocol. Flow cytometry analysis was performed on 2 × 10⁵ cells resuspended in 200 μl of 2% bovine serum albumin (SIGMA) in phosphate buffered saline and labelled for 20 min at 4 °C with 1:200 dilution of fluorescein isothiocyanate (FITC) conjugated anti-CD71 (BD Biosciences; 555536) and 1:200 dilution of phycoerythrin (PE) conjugated anti-CD235a (BD Biosciences; 340947). After staining cells were harvested and resuspended in PBS containing 0.02% Hoechst 33258 pentahydrate nucleic acid stain (Invitrogen). Analysis was performed on an Attune NxT Flow Cytometer (Invitrogen) and subsequently using FlowJo software for gating on viable single cell events and representing the data as a dot plot. Morphological analysis was preformed using a modified Wright stain on a Hemateck 03564647 slide strainer (BAYER). The slides were then imaged on a Nikon inverted microscope.

Spleen and bone marrow cells were collected and homogenized through a 70-µm filter. For the EB analysis, spleen cells were stained with FITC-conjugated anti-CD71 and PE-conjugated anti-TER-119 (BD Biosciences) after homogenization and washing (45 (link), 46 (link)). For the HSPC analysis, bone marrow cells were prepared for staining by erythrocyte lysis (BD Pharm Lyse; BD Biosciences). Cells were then stained in ice-cold PBS containing 2% FBS using the following antibodies: lineage cocktail containing TER-119, CD11b (Mac-1), Ly-6G/Ly-6C (Gr-1), CD3ϵ, and CD45R (B220); CD117 (Kit); Ly-6A/E (Sca-1); CD150 (Slamf1); CD48 (Slamf2); CD16/32; CD34; CD41 (Itga2b); and CD105 (endoglin) (BioLegend and eBiosciences). 4′,6 Diamidino-2-phenylindole (Invitrogen) was used for dead cell discrimination. SLAM and MKEP panels used were described previously (47 (link)–49 (link)). Samples were analyzed by flow cytometry using an LSR II (BD Biosciences). Post-acquisition analysis of data was performed with FlowJo software.