Mepacrine accumulates in structures distinct from lysosomes in platelets. (A-B) Shown are deconvolved, single-plane images from a z-series image stack of 3 individual platelets labeled with each combination individually or together (merge), along with a corresponding differential interference contrast image (DIC). Puncta are labeled by mepacrine (arrows) or by DQ BSA or LysoTracker (arrowheads). The cell outline is based on the DIC image (white dotted line). Scale bars, 1 μm. Platelets were incubated with 50 μM mepacrine (green) and either 10 μg/mL DQ BSA (red; A) or 200 nM LysoTracker Red DND-99 (red; B) for 30 min and analyzed by fluorescence microscopy. (C-D) Quantification of both the degree (mean ± SD) of mepacrine colocalization with DQ BSA or LysoTracker (N = 105 platelets each from 3 individual experiments) and the number of puncta per platelet (mean ± SD) labeled by mepacrine alone (#mep-only), LysoTracker alone (#LyTr-only), DQ BSA alone (#DQ-BSA-only) or by both mepacrine/LysoTracker or mepacrine/DQ BSA (# coloc.). (E) Shown is a deconvolved, single-plane image of a field of platelets labeled with mepacrine (Mep, green) and DQ BSA (red) individually or merged, along with corresponding DIC image. Scale bar, 1 μm. Platelets were analyzed by wide field microscopy at room temperature with a Leica DM IRBE equipped with a 100× Plan Apochromat objective lens (1.4 NA), a Hamamatsu Orca Flash 4 digital CMOS camera, and Leica Application Suite software. Imaging medium was modified, calcium-free Tyrode’s buffer. Deconvolution was performed in Leica Application Suite software using Gold’s iterative deconvolution algorithm. Mepacrine has an excitation/emission of 436/525 nm; LysoTracker Red excitation/emission: 577/590 nm; DQ BSA excitation/emission: ∼590/∼620 nm. Coloc., colocalization; DIC, differential interference contrast; LyTr, LysoTracker; Mep, mepacrine; n/a, not applicable.