Effect on Mitochondrial Membrane Potential and Mitochondrial Activity

Mitochondrial membrane potential (Δψ_m) in C. albicans after exposure to [C₁₆MIM][Cl] was measured using Rhodamine 123 (Rh123) (Sigma, United States) as per Lopes et al. (2013) with minor modifications. Cells were exposed to [C₁₆MIM][Cl] as detailed in the membrane permeabilization experiment. Experiment was conducted in four replicates. The cells exposed to ionic liquid were stained with 20 μM Rh123 for 30 min in the dark. After incubation, excess stain was removed and washed with PBS. Cells were re-suspended in PBS and observed for Rh123 fluorescence under inverted fluorescence microscope (Carl Zeiss, Germany) through FITC filter. Fluorescence of Rh123 was also quantified with 485 nm excitation and 530 nm emission settings using multimode reader (BioTek^®, United States).

MTT assay was used for determining mitochondrial activity of C. albicans cells (Lopes et al., 2013). Cells exposed to [C₁₆MIM][Cl] were harvested, re-suspended in PBS containing 0.5 mg ml^–1 MTT. Cells were incubated at 120 RPM and 37°C for 2 h. End of the incubation, cell suspensions were centrifuged and washed with PBS. Purple formazan product developed from the MTT reduction by mitochondrial dehydrogenases was solubilized in 200 μL DMSO by vigorous vortexing. Suspensions were centrifuged and supernatants were collected for estimating formazan absorbance at 510 nm using UV-Visible spectrophotometer (Shimadzu, Japan). Experiment was performed in duplicates with necessary controls.