Measurement of Membrane Potential in Individual Cells

Membrane potential was measured using BacLight Bacterial Membrane Potential Kit according to manufacturer’s instructions. Briefly, samples were taken from mid-exponential phase (t = 5 h) in S. aureus either grown alone or in the presence of C. albicans. Samples were diluted to 1 × 10⁶ cells in PBS and were stained with DiOC₂(3) for 30 min and analyzed by flow cytometry. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to dissipate membrane potential and was used to gate low membrane potential cells. Bacterial cells were separated from fungal cells and debris using back scatter (BSC) and forward scatter (FSC) parameters with 50,000 events collected for each sample. DiOC₂(3) was excited at 488 nm and emissions of the green and red fluorescence were detected with bandpass filters of 525/50- and 600/60-nm, respectively. Samples were analyzed using FlowJo software. The average and standard deviation was obtained from six biological replicates. Significance was determined using a student’s t-test, P ≤ 0.05.