Dioc2 3

MMP was measured using DiOC2(3) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA) as we described previously [9 (link)]. Briefly, splenocytes were resuspended at 1 × 10⁶/mL in 1 × PBS with 50 nM DiOC2(3) (Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C for 30 min. Cells were washed in 1 × PBS and analyzed by flow cytometry using the 488 nm excitation laser and 530/30 nm bandpass and 670 nm long pass filters. The accumulation of the DiOC2(3) dye [37 (link)] within the mitochondria measured by emission in the green/red channels following excitation reflects the membrane potential. Background corrected mean fluorescence intensity (MFI) using unstained cells was used to calculate the FL3/FL1 ratios of DiOC2(3) test samples as previously described [37 (link)].

The membrane potential of untreated and EGCG-treated planktonic S. mutans was measured using the cationic dye 3,3′-diethyloxacarbocyanine iodide (DiOC₂(3); Molecular Probes, Eugene, OR, USA) by flow cytometry according to the manufacturer’s instructions. DiOC₂(3) exhibits green fluorescence in all bacterial cells, but the fluorescence shifts toward red emission at higher membrane potential values. Briefly, an overnight culture of S. mutans was resuspended in PBS to an OD of 0.3. The bacterial suspension was divided into 1 ml aliquots with different concentrations of EGCG and stained with 10 µl of 3 mM DiOC₂(3) for 30 min in the dark. The samples were analyzed by flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences), using the 488 nm excitation laser and collecting the data using the green (530nm) and red (610/620 nm) filters [40 (link)]. In addition, we measured the forward scatter (FSC) and the side scatter (SSC) of the untreated and EGCG-treated bacteria. FSC detects light scatter along the path of the laser and its intensity is proportional to the diameter of the cell. SSC measures light scatter at a ninety-degree angle relative to the laser and provides information about the granularity of the cell.