Fig. 3
From: Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production
Lack of Opa1 alters mitochondrial morphology and cristae structure. a Transmission electron microscopy (TEM). Primary mature neutrophils from Opa1N∆ and control mice were fixed and analyzed. Representative images are shown. Bars, 5 µm. Insets to the sides, bars, 1 µm. Mitochondria were further analyzed at higher magnification and statistical analyses are provided below in (b), (e), and (f). b The average numbers of mitochondria per neutrophil was quantified in at least 100 cells. Values are means ± SEM (n = 3); n.s., not significant. c Quantitative PCR. Freshly purified mature neutrophils from Opa1N∆ and control mice were analyzed for the number of DNA copies of mitochondrial mouse cytochrome c oxidase subunit 1 (mt-Co1) relative to mouse β2 microglobulin (B2M) which was used as a single copy nuclear-encoded reference gene. Values are means ± SEM (n = 3); n.s., not significant. d Confocal microscopy. Primary mature mouse neutrophils from Opa1N∆ and control mice were stained for TFAM expression (green) and DNA (red) using anti-TFAM antibody and PI. Bars, 10 µm; n = 3. e Average mitochondrial major axis length in freshly purified mature neutrophils from Opa1N∆ and control mice. Images were acquired by TEM and subsequently analyzed using the measurement points module of Imaris software. Data were collected from at least five mitochondria per cell and more than 50 neutrophils per experiment. Values are means ± SEM. ***p < 0.001; n = 3. f Morphometric analysis of cristae width in 60 randomly selected mitochondria of freshly purified mature neutrophils from Opa1N∆ and control mice. Values are lengths in arbitrary units (AU) and shown as means ± SEM. ***p < 0.001; n = 3. g Total cellular ATP production by freshly purified mature neutrophils from Opa1N∆ and control mice was measured by ATP-dependent luciferase activity using an ATP determination kit. Relative luciferase units (RLU) are shown as means ± SEM. **p < 0.01; n = 5. Quantification of the data obtained in activated neutrophils is provided in Supplementary Fig. 6
