Effects of perfusion of NH₄Cl solution on the fluorescence intensity of tPA-GFP before and after opening of the granules. (A) tPA-GFP–expressing cells exposed to 50 mM NH₄Cl solution (dashed box). Representative data on F/F₀ of tPA-GFP secretory granules opened (filled circles, granule i, opened at time “d”) and not opened (open triangles, granule ii) at 1.4 Hz. Top panels: images of whole cell at point d (opening, arrowhead) and f (NH₄Cl exposure). The bar represents 10 μm. Middle panels (a-g): images taken at the times indicated by the arrows from opened (i) and unopened (ii) granules. Brief application of NH₄Cl increased fluorescence intensity of tPA-GFP in unopened granules (i at time “b,” ii at time “f”), but not opened granules (i at time “f”). (B) Percentage increase in relative fluorescence intensity from 111 unopened and 43 opened tPA-GFP–containing granules in 6 cells on NH₄Cl exposure. Five independent experiments are shown as mean plus or minus SD. In the analysis of opened granules, the mean time after granule opening to NH₄Cl exposure was 5.85 minutes.