B29 lsm 510 meta laser confocal microscope

The ROS assay was performed using Image-iT LIVE Green Reactive Oxygen Species Detection Kit (I36007; Invitrogen, Grand Island, NY), according to the manufacturers’ protocol. The cells were cultured in 6-well microwell dishes with coverslips for 48 h and further incubated in medium containing M₄N (80μM) for another 9 to 10 h. After the cells had been washed with warm HBSS/Ca/Mg buffer (Gibco #14025–092; Invitrogen), the cells were incubated in HBSS/Ca/Mg containing 25 μM 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) at 37°C in the dark for 30 min. The cells were then washed very gently with HBSS/Ca/Mg buffer three times. The cells were observed through a B29/Zeiss LSM 510 META laser confocal microscope (Carl Zeiss). The cell images were captured with a 488-nm argon-ion laser, because the oxidation product of carboxy-H₂DCFDA has excitation/emission maxima of approximately 495/529 nm.

The cells were cultured in 6-well microwell dishes and treated with cell culture medium containing JC-1 dye (Cayman Chemical, Ann Arbor, MI) for 30 min, according to the manufacturer’s protocol. After the cells had been washed carefully, they were treated with M₄N for 5 h. The cells were observed through a B29/Zeiss LSM 510 META laser confocal microscope (Carl Zeiss, Jena, Germany). The cell images were captured by two excitation lights, with 488-nm argon-ion and 568-nm argon-krypton lasers. Both JC-1 monomer and J-aggregates are detected by 488-nm excitation light, whereas only J-aggregates are detected by 568-nm excitation light. The ratio of the intensity of the emission light excited by 568-nm light to that excited by 488-nm light in every pixel of an image (the ratio is correlated with the ΔΨ_m) was calculated by the imaging software (Carl Zeiss). The ratio is correlated with the ΔΨ_m. In the figure, the ratio is shown by pseudo-color, where red indicates high ratio (high potential) and dark blue indicates low ratio (low potential). Yellow through green to light blue represents medium ratio (medium potential).