Internalization of IGF-II and insulin through IR-B by flow cytometry. A. HeLa cells expressing IR-B-SYFP labeled in vivo with 50 nM BAC-Ins and 1 nM QD655 at room temperature were incubated at 37°C for different times. Cells were treated or not with acid for 2 min, before collection with 0.5 mM EDTA in PBS and analyzed by flow cytometry detecting SYFP and QD655 signals. QD655 histograms were performed with the population of events which were SYFP positive (transfected cells). Overlays of histograms from cells incubated at 37°C for 90 min and non-incubated cells for each ligand treatment. In each histogram the geometric mean was calculated and was normalized with respect to the time defined as zero. Black asterisks show significant differences with the time defined as zero (p ≤ 0.05; n = 3 independent experiments). Numeral symbol indicates significant differences between acid and non acid treatments (p = 0.01; n = 3 independent experiments). B-C. Similar experiments with BAC-Ins (B) or IGF-II-biot (C). D. In each histogram a marker M1 was determined including approximately 4% of the events at the time defined as zero. The percentage of events inside region M1 was measured and normalized with respect to the time defined as zero (Marker/0 min). The results are shown as the mean ± s.e.m (n = 5 independent experiments). Asterisk indicates significant differences between ligands (p = 0.01).