Jc 10 assay kit

The impact of test NPs on the mitochondrial membrane potential of C. auris was measured using a JC-10 assay kit (Abcam, Cambridge, UK). The experiment was done using the steps given by the manufacturer. The cells (mid-log phase) were subjected to various concentrations of test NPs (0.5 MIC, MIC, and 2 MIC) for 4 h. They were subjected to protoplast preparation, as described previously by Lone et al. [50 (link)]. Then, 90 μL of C. auris protoplasts were mixed with 50 µL JC-10 dye and distributed in different wells of a 96-well microtiter plate (clear bottom-black walled; Thermo Fisher Scientific, Dreieich, Germany) for 1 h in the dark. After that, 50 μL of buffer-B was added to the plate and centrifuged for 2 min at 800 rpm. The readings were captured at Ex/Em = 490/530 nm (X) and 540/590 nm (Y) in microplate readers (Molecular Devices, San Jose, CA, USA). The variation was measured in terms of the Y/X ratio. A decreased ratio confirmed the depolarization of the mitochondrial membrane. Moreover, the experiment included both negative and positive control.

Mitochondrial membrane potential was assessed as instructed by the manufacturer’s protocol (JC-10 assay kit, ab112134, Abcam, Cambridge, Massachusetts, USA) according to the manufacturer’s protocol. Briefly, BeWo cells were plated overnight at 40,000 cells/well in a 96-well plate and subsequently cultured under a normoxic (control) condition (21% O₂, /5% CO₂), or a hypoxic condition (1% O₂ /5% CO₂) for 24 h. Then JC-10 dye-loading solution was added in incubated for 30 min (37°C) and assay buffer was added. The fluorescent intensities for both J-aggregates and monometric forms JC-10 were measured at 515 nm and 570 nm (37°C).

MMP was measured by flow cytometry using a JC-10 Assay Kit (ab112133; Abcam). PBMCs (2.5 × 10⁶/mL)were incubated in nonadherent tubes in a 5% CO₂ incubator at 37 °C with different concentrations of olaparib (0.1, 1, and 10 µM) for 4 h. H₂O₂ (250 µM) was added for the last 2 h. As a positive control for the reaction, a tube was prepared wherein 5 µM of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; Abcam) was added during the last 10 min. The cells were washed with PBS to remove the culture medium and centrifuged at 800× g for 5 min at 23 °C. The supernatant was discarded, and the cells were suspended in 1 mL of sterile PBS and transferred to labeled cytometry tubes. After centrifugation, 500 µL of JC-10 reagent was added to each tube, and the tubes were returned to the CO₂ incubator for another 10 min. The samples were kept at room temperature and protected from light until flow cytometry. The acquisition time limit was 1 h, as per the kit’s recommendation. The intensity ratio of FL1/FL2 was used to monitor the MMP change induced by H₂O₂.

The mitochondrial membrane potential was measured using fluorescence detection (JC-10 Assay Kit, Abcam) according to the manufacturer's instructions. The cells were cultured in 96-well, black-walled, clear-bottom plates and dyed with 50 mL of JC-10 solution. The fluorescence intensities (excitation/emission (Ex/Em) = 485/525 nm and Ex/Em = 540/590 nm) were measured on a multimode microplate reader (Synergy H1 Hybrid, BioTek).

The mitochondrial membrane potentials were analyzed using fluorescence detection (JC-10 Assay Kit, Abcam) according to the manufacturer’s instructions. In brief, the cells were plated onto 96-well, black-walled, clear-bottom plates and dyed with 50 μL of JC-10 solution. The fluorescence intensities (excitation/emission [Ex/Em] = 485/525 nm and Ex/Em = 540/590 nm) were measured on a multi-mode microplate reader (Synergy H1 Hybrid, BioTek). The ratio between aggregate (Em = 590 nm) and monomeric (Em = 525 nm) forms of JC-10 represents the change in the mitochondrial membrane potential. A high ratio indicates a high mitochondrial membrane potential [35 (link)].

Measurement of mitochondrial membrane potential was performed using a JC-10 Assay Kit (Abcam, Cambridge, UK) as previously described (Przepiórska et al. 2023 (link)). JC-10 transpires into mitochondria and polymerizes there in cells with proper mitochondrial membrane potential, forming aggregates with characteristic red fluorescence. Low mitochondrial membrane potential results in a lack of JC-10 transport into mitochondria, leading to JC-10 green fluorescent monomer deposition in the cytoplasm. The cell culture medium was replaced with phosphate-buffered saline (PBS) (Biomed, Poland). After a 15-min incubation, the JC-10 dye solution was added, and the cells were incubated with the reagent for another 30 min. Then, Assay Buffer B was added, and immediate measurements at Ex/Em = 490/525 nm (green fluorescence) and 540/590 (red fluorescence) were performed using the Infinite M200PRO microplate reader (Tecan, Switzerland) and Tecan i-control software. The next step of the analysis was a calculation of the red to green fluorescence ratio. The obtained values were normalized to those of vehicle-treated cells and are expressed as a percent of the control ± SEM.