Figure 3.

Incubation of cells in media at alkaline pH or containing NH₄Cl increases intracellular pH (pHi).A, MEFs were preloaded with cSNARF-1-AM in serum-free DMEM, pH 7.4 (30 min). They were then refed with DMEM/FBS, pH 7.4, and one image set was acquired (preimage #1 pH 7.4). The cells were refed again with the same media, and three image sets were acquired at 40 s, 5 min, and 10 min. At this point, the cells were refed once more with DMEM/FBS at pH 7.4, and another image set was acquired (preimage #2 pH 7.4). Next, the cells were refed with DMEM/FBS at pH 8.3, and three image sets were acquired at 40 s, 5 min, and 10 min. Ratiometric (640:580 nm) pseudocolored images are shown for each treatment condition. Green images reflect the increased pHi (i.e., less-protonated cSNARF1-AM). Graph, quantitation of ratiometric images; n = 100 cells from four fields (∼25 cells/field) ± SD. ∗∗p < 0.01 using one-way ANOVA and Tukey’s post hoc tests. B, MEFs were preloaded with cSNARF-1-AM and treated and analyzed as above, except that cariporide was included during cSNARF1-AM loading (30 min). The cells were then washed once in serum-free media and refed with DMEM/FBS at pH 7.4 or 8.3 without (control) or with cariporide (10 min). Graph, quantitation of ratiometric images as above in panel A. (n = 100 cells). C, MEFs were loaded with cSNARF-1-AM and treated and analyzed as in panel A, except image sets were acquired from cells incubated in DMEM/FBS, pH 7.4, without (preimage) or with NH₄Cl for 40 s, 5 min, or 10 min. Ratiometric (640:580 nm) pseudocolored images are shown for each treatment condition. Graph, quantitation of ratiometric images as above in panel A (n = 100 cells). FBS, fetal bovine serum; MEFs, mouse embryonic fibroblasts; NH₄Cl, ammonium chloride.