Jc 10 assay kit microplate

Change in C. auris mitochondrial membrane potential after exposure to test compounds was detected by using JC-10 assay kit-microplate (abcam, UK), following manufacturer’s instructions. Briefly, 90 μL of C. auris protoplast suspensions and 50 µL JC-10 dye-loading solution was added to the designated wells of clear bottom and black walled 96-well microtiter plates (Thermo Fisher Scientific, Germany). The plates were then incubated for 1 h in dark at 37 °C, followed by the addition of 50 μL of buffer B. After incubation, protoplasts were spanned down at 800 rpm for 2 min. Fluorescence intensity was observed at Ex/Em = 490/530 nm (green fluorescence, represented as X) and 540/590 nm (red fluorescence, represented as Y) with a SpectraMax iD3 multi-mode microplate readers (Molecular Devices, USA). In apoptotic cells, the dye appears green in color as it remains in its monomeric form (Em = 530 nm) whereas, in live cells the dye aggregates in mitochondria and appear red in color (Em = 590 nm). The difference in mitochondrial membrane potential was calculated in terms of ratio of aggregates and monomeric (Y mean/X mean) form of JC-10 dye. Decrease in ratios was interpreted as mitochondrial membrane depolarization. Both negative (untreated cells) and positive (10 mM H₂O₂) controls were considered during experiments.

Mitochondrial membrane potential was assessed with JC-10 Assay Kit-Microplate (Abcam) following general manufacturer's recommendations with some modifications, as detailed elsewhere (32 (link),63 (link)). In brief, ∼2 × 10⁶ cells of each cybrid cell line were harvested, resuspended in 200 μl 1 × JC-10 Assay buffer and then incubated at 37°C for 30 min. Alternatively, harvested cells were preincubated with 10 μM of FCCP for 30 min at 37°C prior to staining with JC-10 dye. After washing with PBS twice, cells were resuspended in 200 μl PBS. The fluorescent intensities for both J-aggregates and monomeric forms of JC-10 were measured at Ex/Em = 490/530 and 490/590 nm with DB-LSR II flow cytometer system (Beckton Dickson, Inc.).