Role of cytosolic anions in lysosome re-acidification after protonophore-induced alkalinization. (A) Schematic representation of the experimental protocol used to dialyze cytosolic anions 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 P2X₇ receptors in the presence of anion substitution buffers (step II). Concurrently, the lysosomal pH was dissipated by treatment with the protonophore FCCP (step II). The plasma membrane was then resealed by removing the P2X₇ agonists and adding divalent cations, while lysosomal re-acidification was initiated by protonophore washout (step III). Lysosomal pH was monitored throughout the protocol. (B) Cells were subjected to the experimental protocol described in (A) while measuring pH. Representative recordings of the fluorescence ratio (proportional to pH) of untreated (filled squares) and CcA-treated cells (open circles) are illustrated (means ± SD of n = 6 and 9, respectively). The stages identified by the Roman numerals correspond to those in (A). Note that inhibition of the V-ATPase with CcA immediately before removal of FCCP fully abrogated lysosomal re-acidification. (C) Cytosolic anion substitution with MeSO₃⁻ did not change the rate or extent of lysosome re-acidification. Representative traces of control (filled squares) and anion-substituted (open circles) cells are shown (means ± SD of n = 6). Summary statistics are presented in Table I.