Fig. 1: Ca2+ signaling requirements of phagosome acidification.
From: Efferocytosis requires periphagosomal Ca2+-signaling and TRPM7-mediated electrical activity
a Preparation of apoptotic cell cargo is schematized. UV-irradiated apoptotic cells are stained with pH-insensitive (CellTrace Violet) and pH-sensitive (CypHer5E) dyes to measure cargo association and acidification, respectively. Apoptotic cells are then offered to macrophages by centrifugation, and unengulfed cells are removed by trypsinization and washes in Ca2+-free saline. b Flow cytometry-based measurement of acidification of phagosomes after engulfment of apoptotic cell cargo by BMDMs loaded with vehicle (DMSO), BAPTA-AM or EGTA-AM (both 10 μM, 30 min). Engulfing CD11b + BMDMs containing labeled cargo (CellTrace Violet) were gated and analyzed for acidification (CypHer5E fluorescence). Bafilomycin A1 (BafA1; 500 nM) and Cytochalasin D (Cyto D; 1 μM) treated cells were used as negative controls. Data points indicate independent samples from different animals; bar charts represent mean value (Error bars = SD). p < 0.001 for all statistical comparisons between treatment conditions by two-way ANOVA (by Time and Treatment [F(4,12) 30.79)] with Bonferroni’s correction). c Representative histograms showing phagosome acidification after 90 min of phagocytosis. Data are representative of biological triplicate samples described in b. d [Ca2+]i dynamics in GCaMP6s-expressing BMDMs during phagocytosis of apoptotic Jurkat cells. GCaMP6s (top image; Fire LUT), CypHer5E fluorescence (middle; gray), and brightfield (bottom image; gray) were acquired using wide-field microscopy (1 frame/7 s). Region of interest (ROI) shown in yellow box is magnified in panel e. White arrows indicate engulfed cell. See also Supplementary Movie 1. e ROI outlined in panel d. is shown as a merged image of GCaMP6s (Fire LUT) and CypHer5E (Cyan) fluorescence, 28 min after the addition of apoptotic cells to BMDMs. Scale bar is 10 μm. f Quantification GCaMP6s (green trace) and CypHer5E (black trace) fluorescence intensity over time. Measurements are from data depicted in panel d over time. Source data are provided as a Source Data file, and statistical testing is described in “Statistics and Reproducibility”.
