Figure 2A, B FreeMitos induce PMN intracellular calcium release in PMN. Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co-incubated with 5 x 106 mitochondria (A). Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN with the vehicle (HBSS) while the dark line represents PMN incubated with mitochondria. PMN intracellular calcium mobilization induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles. PMN microvesicles labeling was performed with either anti-CD66b-FITC labelling (C, left panel) or anti-CD11b-PE (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry panel established to distinguish the PMNL-MVs from the background using the leucocyte specific integrin alpha-M beta-2 (CD11b), the granulocyte specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN were washed twice and resuspended in HBSS before processing on flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC or CD66b-PE. Overlay dot plot of PMNL-MVs population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.E Visual confirmation of PMN and PMN-derived microvesicles. Transmission electron microscopy (TEM) imaging of PMN-derived microvesicles (black arrowhead), representative of 3 biological replicates, from resting or activated PMN (106 cells) incubated with either mitochondria, mitochondrial DAMPs or fMLP as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria (600 nm), DAMPs (600 nm) and fMLP (500 nm) conditions.F Quantification of resting PMN microvesicles produced by PMN (106 cells) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.G Quantification of calpain activity, by fluorescence assay, in cell lysate of either resting (left panel) or activated (right panel) PMN (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.H ELISA quantification of IL-8 concentrations present in the supernatant following the incubation of PMN (106 cells) with freeMitos (107), DAMPs (25 µg) or fMLP (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.