Fig. 3: [Ca²⁺]_ex induces macropinocytosis.

a–f Measurement of calcein uptake by flow cytometric analysis. Freshly isolated human blood monocytes were incubated with LPS and 2.5 mM added [Ca²⁺] for 45 min in calcein-stained (15 µM) RPMI1640/10% FBS containing 5.6 mM [P_i], if not indicated otherwise. Inhibitors were pre-incubated for 30 min in calcein-free medium. 20,000 cells were detected in each experiment. Box-and-Whisker plots show median, 25–75th percentile, and min/max whiskers, of experiments with the indicated numbers of donors. Statistical analysis was performed using two-tailed Mann–Whitney U test b, t-test c, Wilcoxon signed-rank test. d–f p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Representative histogram and analysis strategy for flow cytometric measurement a and quantification of monocytic [Ca²⁺]-dependent calcein uptake in 1 or 5.6 mM [P_i] medium b, quantification of calcein uptake at 37 vs. 4 °C c, after pre-incubation with 2 µM Latrunculin A (Lat A) d, 1 µM Cytochalasin D (Cyto D) e, 10 µM Calhex231 or 10 µM NPS2143 f or the corresponding DMSO concentration prior to [Ca²⁺]-stimulation. g, h Monitoring of CPP formation and their internalization by human monocytes using live-imaging-confocal Raman microspectroscopy (CRM). CRM live cell imaging of one monocyte prior to [Ca²⁺] treatment (0 min) and 60 min after addition of 2.5 mM [Ca²⁺] to RPMI1640 medium (bottom) g. The color-coded images (left) represent the overlay of imaging of cytoplasm, nucleus, lipid droplets, and CPPs. The single images visualize nucleus, cytoplasm, and CPP separately, as indicated. Raman spectra of CPPs in cell-free RPMI medium containing 10% FBS and 2.5 mM [Ca²⁺] (left) and after their internalization into the monocyte (right) h. Characteristic Raman peaks are assigned in the spectra (pyr pyrrole rings of porphyrin, d deformation, s symmetric, as asymmetric). Shown is one cell out of 10 analyzed in experiments with monocytes from four donors.