Jc 10 dye

Changes in the mitochondrial membrane potential (MMP) were assessed with a mitochondrial membrane potential kit using the JC-10 dye (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were seeded into a 96-well black plate. After the indicated treatment, the cells were co-incubated with the JC-10 solution for 1 h at 37 °C according to the kit manufacturer’s instructions. Monomeric JC-10 green fluorescence (λ_ex = 490/λ_em = 525 nm) and aggregate JC-10 red fluorescence (λ_ex = 540/λ_em = 590 nm) were measured using a fluorescence spectrophotometer (SpectraMax M2 microplate reader, Molecular Devices, Sunnyvale, CA, USA), and the ratio of red to green fluorescence was calculated.
For monitoring apoptosis, cells were plated on 18 mm coverslips. After the end of treatment, the fixed cells were reacted with JC-10 solution for 1 h at 37 °C according to the manufacturer’s instructions, and visualized by fluorescence microscopy (ZEISS, Jena, Germany).

To determine the mitochondrial membrane potential using the mitochondrial membrane potential kit (JC-10 dye) (Sigma-Aldrich, St. Louis, MO, USA), the cells (4 × 10³ cells per well) were plated in 96-well black plate and were incubated overnight. The cells were treated with varies dose of SnEA or pyrogallol and incubated for 48 h. After incubation, 100 µL of treated media was removed and added to 50 µL per well of JC-10 dye diluted in assay buffer A, followed by incubation at 37 °C in a CO₂ incubator for 30 min. Then, 500 µL of assay buffer B were added. The fluorescence intensity (λex = 490/λem = 525 nm) and (λex = 540/λem = 590 nm) was monitored for ratio analysis. The ratio of red/green fluorescence intensity is used to determine mitochondrial membrane potential.