Null-point titration determinations of lysosomal K⁺ and Na⁺. Estimates of lysosomal K⁺ and Na⁺ in live RAW macrophages were obtained using the null-point method, as described in Materials and methods. Lysosomes were loaded with a pH-sensitive dextran, and the cytosolic and extracellular compartments equilibrated using bzATP-activated P2X₇ receptors. The lysosomes were alkalinized with the specified amount of TMA (20 mM or 15 mM TMA added where indicated by the filled arrow, and 5 mM TMA where indicated by the open arrow). Changes in lysosome pH upon the addition of a cation/proton exchanging ionophore were measured next to find the null-point, i.e., the condition when addition of the ionophore elicits no change in pH. In the experiments shown in A, nigericin was added to all samples at 0 min (filled arrowhead). The traces are means ± SD of three representative experiments. For each null-point titration experiment, the rate of pH change (DpH/Dt) after addition of the ionophore was measured over 90 s and plotted as a function of the lysosome pH at the time of addition of the ionophore. The data obtained using nigericin and monensin, used to estimate K⁺ and Na⁺ as detailed in Methods, are shown in B and C along with the calculated least-squares linear regressions (R² = 0.9272 and R² = 0.7509 for panels B and C, respectively).