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. 2018 May 9;293(25):9924–9936. doi: 10.1074/jbc.RA118.003166

Figure 3.

Figure 3.

NBC inhibition decreased the concentration of [Na+]i assessed by CoroNa Red fluorescence and flow cytometry. A, FSC and SSC fluorescence data were collected from 20,000 events per sample. Threshold levels for FSC-A and SSC-A were set to exclude signals from cellular debris or abnormal morphology for all samples. B, because BCECF-AM is only incorporated into living cells, BCECF-AM positive cells were used as a control of the viability for CoroNa Red experiments. C and D, cells were loaded with 0.5 μm BCECF-AM and 2 μm CoroNa Red and its fluorescence was evaluated using the FITC (530/30) and PE (575/26) lasers, respectively. Sperm positive for BCECF fluorescence were analyzed for CoroNa Red fluorescence to estimate [Na+]i. E, bright field (BF) and its corresponding CoroNa Red epifluorescence images. F, histogram of percentage of the maximum (% max) versus CoroNa Red fluorescence before and after 5 min addition of 1 μm CCCP. G, histogram of percentage of the maximum (% max) versus CoroNa Red fluorescence before and after the addition of increasing concentrations of ouabain. H, the inhibitor S0859 (5 μm) decreased the concentration of [Na+]i assessed by CoroNa Red fluorescence (n = 3).