S. mutans that has been exposed to the compounds or control bacteria were washed in PBS and incubated with various fluorescent dyes according to the aim of the experiment before being analyzed on flow cytometer. A 20 min staining solution of 3.3 µM SYTO 9 together with 2 µg/mL PI was used to analyze bacteria with disrupted membrane [35 (link)]. This staining was performed at room temperature. For determining the membrane potential, the bacteria were exposed to 15 μM 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)) solution in PBS for 30 min at room temperature prior to flow cytometry [20 (link)]. For determining the membrane and DNA content, the bacteria were exposed to 10 µg/mL Nile red (APExBIO, Houston, TX, USA) and 1 µg/mL DAPI (Sigma) for 30 min at 37 °C [20 (link)]. The following excitation/emission parameters were used: 488 nm/531 nm for SYTO 9 and the green fluorescence of DiOC2(3); 488 nm/620 nm for PI and the red fluorescence of DiOC2(3); 561 nm/635 nm for Nile red and 355 nm/450 nm for DAPI. The samples were analyzed by the LSR-Fortessa flow cytometry instrument (BD Biosciences, San Jose, CA, USA), and 50,000 events were collected using the BD FACSDiva software 6.0. The FCS Express 7 software was used for analyzing the data.
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