Flow cytometry measurement of iron uptake and reactive oxygen species (ROS) generation in RBCs and reticulocytes. Normal peripheral blood cells were diluted (/ml) in PBS, stained with CA-AM or DCF and an APC-conjugated anti-CD71 antibody. Following washing with PBS, they were resuspended in their autologous plasma and then incubated with (+Fe) or without (−Fe) 20 μM FeSO4 for 1 hr. (a) A FSC × CD71 dot-plot, showing the gates for RBCs (CD71−) and reticulocytes (retics) (CD71+). (b) A FSC × CD71 dot-plot of events acquired in the retic gate. (c, d) Histogram distributions of both populations with respect to CA- and DCF-fluorescence, respectively. (e, f) Summaries of the results obtained in each population following incubation with and without Fe. The MFIs and the percent change in the MFIs of each population incubated with and without Fe, calculated per the basal fluorescence (MFI−Fe) are indicated.
