Fig. 7: [Ca²⁺]_ex-induced CPP uptake in monocytes does not induce lysosomal leakage.

a Imaging flow cytometry of acridine orange-stained freshly isolated monocytes from healthy donors (HD) or RA patients after incubation with LPS and either 2.5 mM added [Ca²⁺], 100 µg/ml MSU, or 500 µM LLOMe (10 min) for 4 h. Representative images are shown in left panels, BF brightfield, AO acridine orange, nucleus. Quantification of lysosomal acridine orange mean bright detail fluorescence intensities is shown. b, c Celigo image cytometer analysis of propidium iodide (PI, dead cells) and Hoechst-stained monocytes from 13 donors after stimulation with LPS (n = 13) and either 2.5 mM added [Ca²⁺] (n = 13), 3 mM ATP (n = 12), or CPPs (n = 6) for 16 h at the indicated [P_i] levels b. Whole well scans of Ca²⁺-treated monocytes stained with PI (red)/Hoechst (blue) from one representative donor is shown in b, white framed region is magnified. c Percentage of PI⁺ monocytes of all monocytes (Hoechst⁺). d Measurement of LDH release of monocytes from six healthy donors (HD) and four RA patients after stimulation with LPS and either 2.5 mM added [Ca²⁺] or 3 mM ATP (n = 3) at the indicated [P_i]. LDH release is shown as percentage of total LDH. e Correlation of IL-1β and cell death determined by Celigo in 13 healthy blood donors after stimulation of monocytes with LPS and 2.5 mM added [Ca²⁺] in 5.6 mM [P_i]. Spearman correlation coefficient and level of significance as indicated. f, g LDH release of NLRP3-deficient (f, def-NLRP3) and CaSR-deficient (g, def-CaSR) differentiated THP-1 cells, and their corresponding control cells stimulated with LPS and either 2.5 mM added [Ca²⁺] or 100 µg/ml MSU for 8 h. LDH is shown as percentage of total LDH. h Celigo analysis of PI⁺ cells after stimulation of monocytes with LPS and either 2.5 mM added [Ca²⁺] or MSU for 16 h in the presence of inhibitors of NLRP3 (MCC950), cathepsin B (CA-074-Me), CaSR signaling (Calhex231), or caspase-1 (ZY-VAD-FMK). Box-and-whisker plots show median, 25–75th percentile, and min/max whiskers, bar charts show mean ± s.e.m. Two-tailed Mann–Whitney U test a–d, g, two-tailed independent t-test f, or two-tailed paired t-test h were used. p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.