Snx10 supports the accumulation of high luminal [Cl ⁻ ] within endolysosomes. (A) Schematic of procedure used for [Cl⁻] determinations in sucrosomes. RAW 264.7 macrophages were incubated overnight (16 h) in medium containing 50 mM sucrose, then acutely treated with 3 mM ATP for 30 s in sodium gluconate medium (devoid of Cl⁻) to permeabilize the plasma membrane via the engagement of purinergic receptors, thereby rapidly depleting cytosolic [Cl⁻]. (B) Representative fluorescence images of sucrosome formation in RAW 264.7 macrophages. Cells were pulsed with AlexaFluor-647–conjugated 10 kDa dextran overnight in medium with or without added sucrose, then chased with fresh medium for 1 h prior to imaging. Insets represent times times (3×) zoom images of indicated regions of interest. (C) Cells incubated in the presence of 50 mM sucrose overnight were treated with either vehicle control or 3 mM ATP for 30 s in Na-gluconate medium, then labeled with either FM1–43 dye (top) or cresyl violet (bottom) prior to imaging. (D) Sucrosomal [Cl⁻] was determined colorimetrically as detailed in Materials and methods in WT, CCA-treated (WT + CCA), Cl⁻-substituted for nitrate (WT in NO₃⁻), ClC-7 KO, and Snx10 KO cells prepared as described in A. n = 3. (E) Relative lysosomal [Cl⁻] was determined in WT, CCA-treated (WT + CCA), Cl⁻-substituted for nitrate (WT in NO₃⁻), ClC-7 KO, and Snx10 KO cells using BAC-conjugated 10 kDa dextran as described in Materials and methods. n = 3.