Ti e live cell imaging system

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).

The potential-sensitive fluorescent dye JC-1 was used to determine the MMP. LPS-treated cells and controls were incubated with JC-1 (5 μmol/l) at 37°C for 15 min. JC-1 fluorescence in the cells was observed under an inverted fluorescent microscope (Ti-E Live Cell Imaging System, Nikon).
To visualize the mitochondria, cells were stained with 200 nM MitoTracker Red for 30 min and observed by a confocal microscope (LSM780; Zeiss Microsystems, Jena, Germany).

A cationic dye, JC-1, was used to monitor the MMP. Briefly, cells were treated JC-1 10 μM for 30 min incubation at 37°C. The results were visualized using fluorescent inverted microscope (Nikon, Ti-E Live Cell Imaging System, Japan). Alternatively, fluorescence intensity was monitored using flow cytometry (Becton-Dickinson, FACSCanto II, USA) at excitation/emission maxima of 585/590 nm (J-aggregates) and 514/529 nm (monomer).