Role of lysosomal cations in lysosome re-acidification after GPN-induced alkalinization. (A) Schematic of the experimental protocol used to dialyze and transiently alkalinize lysosomes. After baseline pH measurements in RAW macrophages (step I), the cytosol was dialyzed by selectively permeabilizing the plasma membrane through the activation of resident P2X₇ receptors in the presence of cation substitution buffers (step II). Removal of the P2X₇ agonists and the addition of divalent cations resealed the plasma membrane (step III). The lysosomes were then dialyzed and alkalinized by GPN-induced osmotic swelling (step IV). The lysosomes were next allowed to re-acidify after removal of GPN (step V). An in situ calibration was performed at the end of each experiment (not illustrated). (B) Determinations of fluorescence ratio (proportional to pH) of a representative control experiment where cells were dialyzed with a cytosol-like solution. Each data point represents the ratio data from an individual region of interest. The steps indicated by Roman numerals refer to the protocol schematic of panel A. (C) Histograms of pooled lysosome pH determinations after recovery from the GPN treatment (step V of the illustrated protocol). The median pH of the lysosomes after the re-acidification period is given in the figure along with the total number of lysosomes used to compile the histograms. From left to right: cells dialyzed in cytosol-like (K⁺-rich), Tris-substituted, or NMDG⁺-substituted medium. The rightmost histogram shows cells that were incubated with NMDG⁺-substituted medium but were not permeabilized. The histograms show collated data from nine, three, six, and two separate experiments, respectively.