Figure 6.

CD47 in K562 cells and erythroblasts is sensitive to MiTMAB and this occurs until the orthochromatic erythroblast stage (T120). K562 cells, expanding (EXP) and differentiating erythroblasts (T24, T72 and T120) were incubated with MiTMAB (30 µM) for 15 minutes prior to treatment with Cyt D (5 µM) for 30 minutes at 37 °C. K562 cells were then stained with (A) BRIC32 (anti-CD47) or (B) P5D2 (anti-β₁ integrin) followed by APC-conjugated monoclonal IgG1 anti-mouse secondary antibody, before being assessed by flow cytometry (Average MFI as a % of the DMSO controls ± SD (n = 4)). Erythroblasts were stained with (C) BRIC32 (anti-CD47), (D) P5D2 (anti-β₁ integrin) or (E) BRIC71 (anti-band 3) followed by APC-conjugated monoclonal IgG1 anti-mouse secondary antibody before being assessed by flow cytometry (Average MFI ± SEM (n = 2); ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 using the Students T test). Emissions from 10,000 events were detected using a MACSQuant Analyser 10 flow cytometer and data was analysed using FlowJo version 10.