Figure 3.

Raising extracellular pH increased reactive oxygen species (ROS) production. Neutrophils preloaded with DHR123 ROS indicator dye in media of various pH values were activated with media (−ve control) or phorbol myristate acetate (PMA). The ROS generation kinetics was estimated by a fluorescence plate reader up to 30 min post activation. (A,B) The R123-based ROS generation kinetics showed that elevating pH increased ROS production in both control and PMA-treated neutrophils. As shown in the inset regression plot, the magnitude and the rate of ROS production were higher for PMA-treated neutrophils than control neutrophils (n = 3–4; *p < 0.05, between pH 6.6 and 7.8 conditions at respective time points; two-way ANOVA with Bonferroni’s posttest conducted at each time point; best fit non-linear polynomial second-order regression analysis; p-value in each inset graph shows whether the slope is different than 0; error bars represent SEM). (C) The ROS generation in neutrophils activated with either control or PMA at pH 6.6, 7.4, or 7.8 was imaged by confocal microscopy. The R123 (green) and DNA (blue) fluorescence staining at 30 min showed more ROS at higher pH in both control and PMA-activated neutrophils, although the amount was greater in PMA-treated cells (n = 3; scale bar 20 μm). (D) Flow cytometry analyses were performed to detect the ROS production in each cell. DHR123-preloaded neutrophils were activated either by media (−ve control) or PMA for 30 min at different pH conditions (pH 6.6, 7.4, and pH 7.8). Mean fluorescence intensities (percentage of maximum) showed higher ROS production at higher pH (n = 3; *p < 0.05, between pH 6.6 and 7.8 conditions; one-way ANOVA with Dunnett’s posttest).