Mepacrine labeling is less sensitive to deacidification than LysoTracker labeling in platelets. (A) Platelets were pretreated with vehicle or with bafA1 and then labeled with mepacrine (green) and LysoTracker Red DND-99 (red) together with vehicle or 100 nM bafA1 for 30 min. Shown is a single-plane image of a single platelet from each treatment, showing each label alone or merged (Mep/LyTr) and the corresponding DIC image; the middle and lower panels are raw images, and the upper panels are deconvolved images of the middle panels. The cell outline is based on the DIC image (dotted line). Scale bar, 1 μm. (B) Quantification of fluorescence intensities of mepacrine or LysoTracker Red in control or bafA1-treated cells (taken from raw images), expressed as a percentage of the untreated control (mean ± SD). N = 105 platelets from 3 separate experiments. 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 CMOS camera, and Leica Application Suite software. Imaging medium was modified, calcium-free Tyrode’s buffer. Deconvolution was performed with Leica Application Suite software using 3 iterations of Gold’s iterative deconvolution algorithm. Mepacrine has an excitation/emission of 436/525 nm; LysoTracker Red excitation/emission: 577/590 nm. ****P < .0001.
