Anti human gpa pe cd235a hir2

TF-1 cells were induced with DOX for 48 hours and then differentiation was induced with EPO as previously described (Wang et al., 2013 (link)). Surface expression of GPA was analyzed using anti-human GPA-PE (CD235a) (HIR2, BD Biosciences). Expression of HBG1/2 and KLF-1 was analyzed by qRT-PCR.
For the DOX washout experiment, cells were washed 5 times in PBS and then resuspended in medium supplemented with EPO for an additional 4 days. The cells were then analyzed for surface expression of GPA and expression of HBG1/2 and KLF-1 as described above.

Primary human CD34-enriched cord blood was transduced with lentivirus (pLVX EF1α-IRES-zsGreen; Clontech) overnight, and sorted for GFP expression using a FACSAria II (Becton-Dickinson). For progenitor media assay, cells were, washed and incubated in HPGM medium (Lonza) supplemented with FLT3L, thrombopoietin and stem cell factor (SCF). The cytokines were all purchased from Peprotech and used at 20 ng/ml. 6 days post-culture, differentiation of GFP-positive cells was assessed by flow cytometry using anti-human CD34-APC (8G12) and CD38-PE-Cy7 (HB7) (BD Biosciences). For myeloid differentiation assay, cells were cultured in Myelocult H5100 (Stem Cell Technologies) with 20-µg/mL each of IL-3, SCF, FLT3L, and GM-CSF (Peprotech) for 6 days. Myeloid differentiation was assessed by flow cytometry using anti-human CD33-PE (WM53) and CD14-APC-Cy7 (MφP9) (BD Biosciences). For erythroid differentiation assay, cells were cultured in Stemspan II with erythroid expansion kit (Stemcell Technologies) for 6 days. Erythroid differentiation was assessed by flow cytometry using anti-human GPA-PE (CD235a) (HIR2) and CD71-PE-Cy7 (OKT9) (BD Biosciences).