Fig. 1: Intracellular ROS induced by mefloquine are localized inside the mast cell granules.
From: Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production
a Representative live confocal images illustrating the granule damage and production of ROS in mefloquine-treated mast cells. BAM-anchored bone marrow-derived mast cells (BMMCs) were pre-incubated with LysoTracker Red DND-99 (to label granules) and CellROX Deep Red (green; to monitor ROS production) for 30 min at 37 °C. Cells were then washed and live confocal imaging was performed immediately. After an initial image recording for 72 min (t = −72), mefloquine (20 μM) was added to the cells (t 0) and image recording continued for 112 min (t = 112). Scale bar = 5 µm. b Kinetics of LysoTracker and CellROX signal intensities in mefloquine-treated BMMCs, representing the intensity from 800–1000 cells in total. c Representative live confocal images illustrating intracellular localization of ROS (CellROX signal in green) within mast cell secretory granules (LysoTracker signal in red) upon mefloquine treatment. Intensities at each time point have been normalized to accentuate the localization of signal. Scale bar = 5 µm. d Quantification of the colocalization of CellROX with LysoTracker. Colocalization analysis was carried out on single confocal slices from four independent full fields of view at the indicated time points, representing 800–1000 cells in total. Bars indicate mean Mander’s M1 coefficient ± SEM (n = 4), one-way ANOVA with Dunnett’s multiple comparisons test. e GSH/GSSG ratio in response to mefloquine treatment. BMMCs were incubated ± mefloquine (20 µM) for 30 min. Cell lysates were prepared and incubated with Luciferin Generation Reagent (30 min) followed by incubation with Luciferin Detection Reagent (15 min). Subsequently, luminescence was measured using a TECAN microplate reader and the GSH/GSSG ratio was determined. Mean ± SEM (n = 3), unpaired, two-tailed Student’s t-test. f ROS levels in wild type (WT) and serglycin-deficient (SG−/−) mast cells. BMMCs were preincubated ± 8 mM N-acetylcysteine (NAC) for 2 h followed by incubation ± 20 μM mefloquine (MEF). After 30 min, cells were washed and incubated (30 min) with CM-H2DCFDA. Cells were then washed and the fluorescence intensity was immediately assessed by flow cytometry. The bars represent means ± SEM of the geometric mean fluorescence intensity (gMFI) for CM-H2DCFDA (n = 4)
