Spectrofluorometer

Mitochondrial membrane potential reflects the functional state of the mitochondria within cells (Wadia et al., 1998 (link)). Changes in MMP were measured by the uptake of 5, 50, 6, 60-tetrachloro-1, 10, 3, 30-tetraethylbenzimidazolcarbocyanine iodide (JC-1) into the mitochondria. When excited at 488 nm, the monomeric form of JC-1 has an emission maximum at 525 nm, but the aggregated form (J-aggregates) has an emission maximum at 595 nm (Reers et al., 1991 (link)). Cells and isolated mitochondria were incubated with JC-1 at 37°C for 30 min in medium or reaction buffer (0.32 mmol/L sucrose, 10 mmol/L Tris, 20 mmol/L Mops, 50 μmol/L EGTA, 0.5 mmol/L MgCl₂, 0.1 mmol/L Pi (K⁺), 5 mmol/L sodium succinate in the presence of 5 μg/mL JC-1). At the end of the incubation, the dye-loaded cells and mitochondria were collected by centrifugation, washed extensively with reaction buffer to remove excess dye, and then resuspended in the same buffer at the appropriate dilution. The cells were visualized under an inverted fluorescence microscope (Nikon, Ti-E Live Cell Imaging System Japan), and the fluorescence intensity was measured (488 nm excitation and 595 nm emission) on a Molecular Device spectrofluorometer (United States).

Reactive oxygen species (ROS) levels were measured using the ROS-specific probe 5′,6′-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Beyotime Institute of Biotechnology, Nantong, China). Cells and mitochondria were incubated with 10 μmol/L H2-DCFDA for 30 min at 37°C in medium or reaction buffer (0.32 mmol/L sucrose, 10 mmol/L Tris, 20 mmol/L Mops, 50 μmol/L EGTA, 0.5 mmol/L MgCl₂, 0.1 mmol/L Pi (K⁺), 5 mmol/L sodium succinate) (Friberg et al., 2002 (link)). Next, the cells were visualized under an inverted fluorescence microscope (Nikon, Ti-E Live Cell Imaging System, Japan), and the mitochondria were monitored kinetically for 60 min at 37°C on a Molecular Device spectrofluorometer (United States) with 488 nm excitation and 525 nm emission filters.

Caspase-8 can induce classic apoptosis, which involves the activation of caspases that are associated with the assembly of cytoplasmic DEFs (Siegel et al., 1998 (link)). The activities of caspase-8 and caspase-3 were measured using IETD-AFC and DEVD-AFC (BioVision). Cells were collected from monolayers and then lysed in 60 μl of cell lysis buffer for 10 min on ice. After centrifugation, 45 μl of supernatant were extracted and added to a 96-well plate. Next, 50 μl of 2 × reaction buffer (containing 10 Mm dithiothreitol) were added to each sample. Finally, 5 μl of the 1 Mm IETE-AFC or DEVD-AFC substrates (50 μM final concentration) were added, and the mixture was incubated for 1–2 h at 37°C. Levels of cleaved caspase substrate were measured using a spectrofluorometer (Molecular Devices).

Levels of cell-free DNA in culture medium were measured using the Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Invitrogen, Carlsbad, CA, USA). Supernatants from cultured neutrophils were collected, mixed with assay kit reagent, and fluorescence was measured using a spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at excitation/emission of 480/540 nm. DNA concentrations were calculated using a standard curve prepared using kit-supplied DNA. Data were normalized relative to NET-DNA (ng/mL) released by stimulating cells with ATP, BzATP, or PMA.