Jc 1 dye

MPP was assessed using the lipophilic cationic probe JC-1, which is a sensitive fluorescent dye (Invitrogen, USA). Briefly, after incubating as described above, cells were incubated with 5 mM JC-1 dye (Molecular Probes) at 37 °C for 15 min. Cells were then washed three times with PBS and immediately analyzed with a fluorescence microscope (Olympus, Japan). Red emission indicated membrane potential-dependent JC-1 aggregates in the mitochondria. Green fluorescence indicated the monomeric form of JC-1 entering the cytoplasm after mitochondrial membrane depolarization. The ratio of red to green fluorescence intensity was measured and data were expressed as relative to the mean sham value.

The CM-H2DCFDA dye, Fluo-4 AM calcium dye (Molecular Probes, United States), and JC-1 dye (Molecular Probes, United States) were employed to assess ROS, calcium, and MMP, respectively. The fluorescent images of calcium and JC-1 were captured by a confocal microscope. H9c2 cells (1 × 10⁴/well) were seeded in μ-Slide 8-well glass bottom plates. Cells were labeled by Fluo-4 AM or JC-1 probe for 30 min after LS102 treatment according to the manufacturer’s protocols. The fluorescent intensity of images was analyzed by using the Image J software and a randomly captured method was used to make sure there were three fields per group. The fluorescence intensity of calcium, ROS were detected by the flow cytometer. All cells in the 6-well plates were collected and then suspended in a pre-warmed DMEM buffer containing CM-H2DCFDA or Fluo-4 AM calcium dye. Each group (1 × 10⁴ cells) intensity was calculated as representation.

Apoptosis in PC12 cells was analyzed through the measurement of mitochondrial membrane potential quantification by flow cytometry using JC-1 dye (Molecular Probes). After incubation with 6-OHDA, CCCP (carbonyl cyanide m-chlorophenylhydrazone, positive control) and QP water extract, PC12 cells were collected and centrifuged at 1200 rpm for 5 min. Removing the supernatants, the cell pellets were washed twice and resuspended in 500 μL cold PBS. Then the cell suspension was transferred to 1.5 mL microcentrifuge tubes and centrifuged at 1200 rpm for 3 min. Aspirating the supernatant, cell pellets were resuspended in 500 μL warm PBS. Then, 4 mM JC-1 dye was added to each sample to make the final concentration as 15 μM before incubation at 37°C for 20 min. The proportion of aggregated versus monomeric JC-1 probe was quantified using the ratio of fluorescence emissions at 590 nm (red) over 530 nm (green) with a FACSCalibur flow cytometer. Consequently, the mitochondrial depolarization, which means the loss in mitochondrial membrane potential, is indicated by a decrease in the red/green fluorescence intensity ratio.

Mitochondrial membrane polarization was measured using tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) dye (Invitrogen/Molecular Probes, Eugene, OR, USA). hTCEpi cells were cultured in growth media supplemented with or without high glucose or mannitol for 14 days. Cells were then seeded onto 35 mm glass-bottom dishes (MatTek Corporation, Ashland, MA, USA) and allowed to adhere overnight. Cells were then incubated at 37 °C with 10 μg/mL of JC-1. After incubation, cells were washed three times in PBS and imaged on a Leica SP8 laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) using a 63× oil objective. The microscope was enclosed within an environmental chamber that maintained cells at 5% CO₂ and 37 °C during imaging. J-aggregates (multimers) were scanned using a 488 nm excitation laser. J-monomers, indicating mitochondrial polarization, were scanned using a 568 nm laser. All images were sequentially scanned to avoid spectral crosstalk between channels. The entire experiment was repeated two additional times.

Oxalate, C-phycocyanin (CP), DMEM (Dulbecco's Modified Eagle Medium), fetal bovine serum (FBS), propidium iodide (PI), trypan blue, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide, trypsin, Tween 20, and Triton X-100 were purchased from Sigma Chemicals (St. Louis, MO, USA). 2, 7–dichlorofluorescein diacetate (DCF-DA) (Molecular probes, Inc. USA), and JC-1 dye (Molecular probes, Inc. USA) were procured from Molecular Probes, Inc. (USA), P-JNK/SAPK and P- ERK1/2 antibodies from Cell signaling Technology, USA. T-JNK and T-ERK1/2 antibodies were purchased from Santa Cruz Biotechnology, INC, USA.

The MMP was checked using the JC1 dye (Molecular Probes, Eugene, OR, USA) (Librizzi et al., 2012[31 (link)]), which exhibits potential-dependent accumulation in intact mitochondria where the compound undergoes a fluorescence emission shift from green (~529 nm) to red (~590 nm). Thus, in the case of dissipation of MMP, a decrease in the red/green fluorescence intensity ratio can be observed. As a positive control, cells were treated with 1 µM valinomycin, a mitochondria-depolarizing K⁺ ionophore.

Changes in ΔΨ_m were assayed using the mitochondrial fluorescent dye JC-1. B16F10 cells were seeded at a starting density of 2.5 × 10⁴ cells/well in 6-well plates containing coverslips and cultured at least overnight before the experiment. After subjecting the cells to different concentrations of DEA for 3 h, the coverslips were rinsed twice in PBS and placed upside down on top of a small chamber formed by a microscope slide filled with PBS supplemented with 10% FCS and 1 μg/mL of JC-1 dye (Molecular Probes, Eugene, OR, USA). The cells were imaged with a Nikon Eclipse Ti-U fluorescent microscope (Auro-Science Consulting Ltd., Budapest, Hungary) equipped with a Spot RT3 camera, using a 20× objective lens with epifluorescence illumination. After the cells were loaded with dye for 15 min, the same microscopic field was imaged with a 490 nm bandpass excitation and > 590 nm (red) and < 546 nm (green) emission filters. Under these conditions, we did not observe considerable bleed-through between the red and green channels. The quantification of JC-1 fluorescence intensities in each sample was performed in 15 randomly chosen microscopic fields containing 20–30 cells using the MetaXpress image analyzer software (Molecular Devices LLC., San Jose, CA, USA). These experiments were repeated three times.