Distinguishing the internalized ITs from those bound on the cell surface by using quenching effect of Trypan Blue (TB). All the flow cytometry and microscopic experiments are in the presence of sCD4-183 (300 ng/ml) (A) Before incubation with ITs, the viability of cells was 96%. The diagrams show percentage of 7B2-PAC-Alexa488 that remained fluorescent after the addition an increasing concentration of TB (1, 2 and 3 mg/ml). In the presence of NaAz, 7B2-PAC can only attach to the cell membrane, without cell internalization. As the right diagram demonstrates, the addition of an increasing concentration of TB shows that a concentration of 3 mg/ml of TB is sufficient to quench the extracellular fluorescence. In the absence of NaAz, due to the IT internalization, the fluorescence intensity of ITs remain intact from quenching by TB (B,C). Dot plots of cells incubated with 7B2-PAC-Alexa488 (B) or 7B2-RAC-Alexa488 (C) in the absence of NaAz, analyzed by flow cytometry before TB addition and 2 hr after that. Before TB addition, the dot plots of the cell population incubated with Alexa-ITs, either adherent to plasma membrane or internalized, are emitting green fluorescence (FL1). TB cannot enter the live cells, therefore, after TB addition the green fluorescence emission (FL1) is not quenched, while we observe the upshift of cell population corresponding to the cells with damaged membrane (ie. 4% dead cells) and Alexa-ITs adherent to the membrane which emitted red fluorescence (FL3) after quenching. Results are representative of two independent experiments. (D) Live confocal microscopy by taking images from a series of different regions started with time after the addition of Abs, generally 50 observations during the 90 min period. Live cells incubated with 7B2-PAC-Alexa488 demonstrate the presence of IT on the cell surface after 15 min. Following 90 min, 7B2-PAC is observable both internalized and on the cell surface. By imaging the same region, after 5 min TB addition, only the green fluorescence on the cell surface is quenched and emitting red fluorescence (shown by white arrow). The green and red fluorescences are detected by band-pass filters 530 ± 30 nm and 650 ± 13 nm, respectively.