Analysis of TfR binding and internalization using Amnis flow imaging cytometry. (A) CD71/Ter-119 colocalization imaging of erythroblasts obtained from the bone marrow of WT, RBC6 (Tfrc^+/L645R), and RBC21 (Tfrc^+/R654H) mice. Intensity of CD71/Ter-119 overlap (yellow) generates the median bright detail similarity score. (B) Tf/CD71 colocalization imaging of erythroblasts from the 3 cohorts. Intensity of Tf/CD71 overlap (pale blue) generates the median bright detail similarity score, which is normalized to generate the mean percentage of Tf saturation. (C) Tf/Ter-119 internalization imaging of erythroblasts from each cohort at 0 minutes (before initiation of endocytosis). The mean internalization erode score of Tf uses Ter-119 as the mask. (D) Tf/Ter-119 internalization imaging of erythroblasts from each cohort at 5 minutes postinitiation of endocytosis. All scores represent 2000 Ter-119⁺ events captured in each cohort. (E) Total time course of Tf uptake, via endocytosis, calculated in Ter-119⁺ erythroblasts from each cohort. Mean ± (SD) shown in error bars. (F) Quantitative real-time polymerase chain reaction of liver Hamp-1 messenger RNA expression in each cohort. Mean ± (SD) shown (n = 6). *P < .05, **P < .01. MAD, median average deviation.