Co-detection of TfR-SEP and TfR-pHuji within the same endocytic vesicle. (A) Portion of a 3T3 cell cotransfected with TfR-SEP and TfR-pHuji. (B) Fluorescence in the green and red channels of segmented clusters at pH 7.4 in a representative cell. Yellow dots, clusters detected in both channels (422 clusters); green dots, clusters detected in the SEP channel only (no matching structure in the red channel, 38 clusters); red dots, clusters detected in the pHuji channel only (no matching structure in the green channel, 89 clusters). Black dashed line shows linear regression of all TfR-SEP clusters (R = 0.85). Green and red lines show the lower detection limits of SEP and pHuji clusters, respectively. (C, a) Proportion for n = 5 cells of clusters detected in both channels (yellow) or with either TfR-pHuji (red) or TfR-SEP (green) as shown in B. (b) TfR-SEP clusters enriched in TfR-pHuji (yellow) or not (green) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and red). (D) Example of a scission event co-detected with TfR-SEP and TfR-pHuji. (E) Same analysis as in B performed on the fluorescence of CCVs at their time of detection at pH 5.0. Yellow dots, 303 CCVs; green dots, 570 CCVs; red dots, 119 CCVs. Black dashed line shows linear regression of all TfR-SEP–detected scission events (R = 0.59). Green and red lines show the lower detection limits of SEP and pHuji CCVs, respectively. (F) Same analysis as in C performed on detected CCVs at pH 5.0. SEP and pHuji lower detection limits were determined as in E. (G) Mean TfR-SEP and TfR-pHuji fluorescence at pH 7.4 (top) and 5.0 (bottom) of scission events detected in the green channel (3,721 events in five cells) aligned to the time of CCV detection. The black lines indicate 95% confidence intervals for significant enrichment. (H) Same as G with 2,177 scission events detected in the red channel. Error bars represent SEM.