Lysosomal H ⁺ leak is extremely small and is unlikely mediated by ion channels including TMEM175. (A) Experimental procedure for measuring lysosomal H⁺ leak. Cells are loaded with OG followed by chase with fresh culture medium for 3 h (hippocampal neurons) or 2 h (peritoneal macrophages). The lysosomal pH is imaged before and continuously after V-ATPase blocker, concanamycin A (ConcA), is added to the cells (5 μM for neurons and 2 μM for macrophages) as well as after a weak base, NH₄Cl, is added to measure buffering capacity, followed by an in situ calibration for each coverslip. (B) Representative lysosomal pH changes of a cultured WT and TMEM175 KO hippocampal neuron in response to ConcA and NH₄Cl. (C and D) Summaries of the various parameters extracted from the experiment in hippocampal neurons (C) and macrophages (D) for WT and TMEM175 KO mice. There is no statistical significance between WT and KO cells in pH change over time, buffering capacity, or [H⁺] decrease rate with an unpaired two-tailed t test. Each circle represents one cell with n = 12 for WT and KO hippocampal neurons and n = 9 for WT and n = 10 for KO macrophages. Hippocampal neurons (C) are from three TMEM175 KO lines (K1, K3, and K6, as indicated in Fig. 5 legend), while macrophages (D) are from two TMEM175 KO lines (K1 and K3). H⁺ decrease rate (in mM/min) was calculated as the product between pH change rate (pH/min) and buffering capacity (mM/pH) for each cell. (E) Table of values from key parameters summarized in the previous bar graphs (C and D). * For total H⁺ leak of a lysosome, an assumption was made that lysosomes are spheres of 300-nm diameter and the calculated H⁺-leak values are based on the average [H⁺] decrease presented in the table. Error bars are SEM.