Jc 1 mitochondrial membrane potential detection kit

The mitochondrial membrane potential was measured using the JC-1 mitochondrial membrane potential detection kit (#30001, Biotium Inc., USA), as previously described [15 (link)]. Cells were seeded in six-well dishes containing cover glasses at a density of 1 × 10⁵ cells per well. After incubation, the cells were treated with CQ (10^-4 M) alone for 6 h; LE (Lipofundin MCT/LCT, 0.75%) or ROS scavenger (10^-4 M NAC or 10^-5 M mitotempo) for 1 h, followed by CQ (10^-4 M) for 6 h; or LE (0.75%) or ROS scavenger (10^-4 M NAC or 10^-5 M mitotempo) alone for 7 h. The cells were washed with PBS and then, 1 × JC-1 dye reagent working solution was added to the cells for 15 min, and the cells were washed with PBS. The mitochondrial membrane potential was analyzed using a fluorescence microscope (Nikon Eclipse Ti2) and calculated as the fluorescence intensity of the JC-1 red/green ratio and normalized to the control.

HTR-8/SVneo cells in six-well plates were treated with Cu-NPs (10, 20, or 40 mg/ml) for 12 h. The mitochondrial membrane potential (MMP) was monitored using the JC-1 mitochondrial membrane potential detection kit (Biotium, Hayward, CA, USA). In healthy cells, JC-1 (a fluorescent cationic dye) enters the negatively charged mitochondria, where the dye aggregates and fluoresces red. In cells in which the MMP has collapsed, JC-1 exists as monomers throughout the cell, which fluoresce green. Consequently, mitochondrial depolarization is indicated by a decrease in the ratio of the red:green fluorescence intensities. Data acquisition was performed using a MACSQuant Analyzer BD-FACS Canto II (Miltenyi Biotec, Becton-Dickinson, San Jose, CA, USA), and the data were analyzed using FlowJo 7.6 software (Tree Star, Inc., Ashland, MA, USA).

Loss of ΔΨm, a hallmark for early apoptosis, was measured using the JC-1 mitochondrial membrane potential detection kit (Biotium, Hayward, CA). JC-1 contains a cationic dye (5,5’,6 (link)6’-tetrachloro-1,1’,3 (link)3’-tetraethyl-benzimidazolyl- carbocyanine-iodide) that fluoresces red within the mitochondria of alive cells. In the dying cells, the mitochondrial membrane potential collapses and the cationic dye remains in the cytoplasm but fluoresces green. In healthy cells, the red to green fluorescence ratio is higher compared to the cells entering apoptosis.
We performed the JC-1 assay as per the supplier's instructions. Briefly, after 24 h 2-EP exposure, the cells were rinsed with fresh media and incubated for 15 min with 500 μl/well of JC-1 reagent in culture media. Images were captured using a fluorescence image scanner (FMBIO III; Hitachi) and the red/green fluorescence ratios were calculated.

Mitochondrial membrane potential was monitored using JC-1 mitochondrial membrane potential detection kit (Biotium Inc., Hayward, CA, USA) according to the manufacturer's protocol. Primary cultures of hippocampal neurons from G17.5 fetal brain were grown and treated with ethanol and osmotin as described earlier. After 24 h of drug treatment, cells in triplicate culture plates were harvested, stained with JC-1 reagents at 37°C for 15 min, washed twice in 1 × assay buffer, and resuspended in 0.5 ml PBS for FACS analysis (FACSCalibur Flow Cytometer; Becton Dickinson, San Jose, CA, USA). JC-1 aggregates in healthy polarized mitochondria emit red fluorescence at 590 nm. JC-1 monomers that leak from stressed depolarized mitochondria emit green fluorescence at 530 nm.^{51 (link)} The red and green fluorescence was measured in the Green (FL-1) and Red (FL-2) channel of the flow cytometer, respectively. The cells were then immediately observed with a fluorescence microscope using a ‘dual-band pass' filter designed to simultaneously detect fluorescein and rhodamine or fluorescein and Texas Red (Hayward, CA, USA).