Detection of endocytic vesicles containing TfR-red FPs. (A) Images of NIH-3T3 cells transfected with TfR fused to SEP, pHTomato, pHoran4, or pHuji at pH 7.4. (B) Details corresponding to the boxed areas in A, at pH 7.4 (left) and 2 s later at pH 5.0 (middle, same contrast; right, increased contrast as indicated). Note the complete quenching of some clusters (blue arrowheads), whereas others are still visible (yellow arrowheads) at pH 5.0. (C) Examples of events detected in cells transfected with the four FPs. (D) Mean frequency of scission events detected with the different markers. The number of cells tested is indicated. (E) Proportion of terminal scission events with the different markers. (F) Mean fluorescence of nonterminal (dark green, 984 events) and terminal (light green, 802 events) scission events at pH 7.4 (top) and 5.0 (bottom) aligned to their time of detection in 14 cells transfected with TfR-SEP. The black lines indicate 95% confidence intervals for significant enrichment. (G) Same as F for eight cells transfected with TfR-pHuji (dark red, 598 nonterminal events; light red, 447 terminal events). Error bars represent SEM.