Jc 10 kit

The mitochondrial membrane potential assay was carried out using a JC-10 kit (Abcam, CA, USA) [31 (link)]. SH-SY5Y cells were seeded in 96-well plates and pretreated with various concentrations of GH (dissolved in PBS). After incubation for 1 h at 37°C, scopolamine was cotreated with GH for 24 h at 37°C. After treatment, the cell mitochondria were stained using 50 μL of JC-10 solution for 30 min at 37°C and kept away from light. After incubation, 50 μL of buffer B solution was added into the JC-10 loading plate before reading the fluorescence intensity, and this was analyzed using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The fluorescence intensities were measured at Ex/Em = 490/525 nm and 490/590 nm for ratio analysis.

A total of 10,000 cells were seeded in each well of black opaque cell plates and treated with either 2 mg/ml of gentamycin or PBS as a control for 24 hours. Then, following the manufacturer’s instructions, treatment with the JC-10 kit (Abcam, ab112134) was performed as follows: 50 µl of JC-10 was added per well, followed by incubation for 1 hour before adding buffer B. Fluorescence was subsequently recorded using a Synergy H1 (BioTek) at an excitation wavelength of 488 nm and emission wavelengths of 529 nm and 590 nm. The 590 nm vs. 529 nm ratio served as the relative membrane potential.

The effect of the most active compound C5 on C. auris mitochondrial membrane potential (MMP Δψm) was quantified by using the JC-10 kit (Abcam, UK), according to the directions of manufacture. Briefly, 90 μL of the prepared protoplasts were added with 50 μL JC-10 dye into vibrant bottom and blackened walled 96-well plates and left in dark at room temperature for 1 hour. After incubation period, 50 μL volume of kit’s buffer-B was added, and then microtiter plate was rotated at 800g for 2 minutes. The excitation-emission maxima ratio (Ex/Em = 490/530nm and 540/590nm) was calculated using a microplate reader (Spectra-Max iD-3 multi-mode, USA). The fluorescence intensity (green fluorescence) referred to as X was calculated by using Ex/Em = 490/530nm, while the red fluorescence referred to as Y was calculated by using Ex/Em = 540/590nm. The reduction in mitochondrial membrane potential (MMP) in exposed cells was measured using the aggregate/monomeric (Y-mean/X-mean) ratio of JC10 dye. The decrease in ratio was regarded as depolarization of the mitochondrial membrane. During the tests, negative (untreated cells) and positive (treated with 10 mM H₂O₂) controls were also included.