Secretory kinetics of tPA-GFP determined by TIR-F microscopy. (A) Secretory kinetics of tPA-GFP in EA.hy926 cells analyzed by TIR-F microscopy using recording medium containing 50 μg/mL TRITC-conjugated anti-GFP antibody. Changes in the fluorescence intensities of both GFP and TRITC were monitored simultaneously through W-View system at 1.4 Hz. The means plus or minus SD of relative changes in fluorescence intensity (F/F0) of tPA-GFP (●) and TRITC-conjugated anti-GFP antibody (△) in 72 granules from 9 cells tested are shown in panel A (5 independent experiments). Sudden increase in green fluorescence (▲) was followed by a gradual increase in red fluorescence. (B) Individual TF1/2 (the time required for the fluorescence to decline to half of its peak value), which defines the half-life of exocytosed tPA-GFP disappearance from the membrane surface, are plotted (77 granules in 10 cells). Expanded time points within 50-second TF1/2 are shown in the inset. (C) Number of total occurrences calculated for individual TF1/2 (abscissa) shown as percentages of total occurrences (ordinate).
