Baclight membrane potential kit

The immediate effect of CBD on the membrane potential of S. mutans was analyzed using the BacLight Membrane Potential Kit (Molecular Probes, Life Technologies, Eugene, OR, USA) according to the manufacturer’s instructions. Overnight S. mutans cultures were resuspended in PBS at an OD_600nm of 0.3 and exposed to various concentrations of CBD, and immediately thereafter, 3,3′-diethyloxacarbocyanine iodide (DiOC₂(3)) was added to a final concentration of 30 μM. After 30 min the bacteria were analyzed by flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences) using the 488 nm excitation laser and collecting the data using the green (530 nm) and red (610/620 nm) filters [49 (link)]. The BD FACSDiva software was used for the collection of data and the FCS Express 7 software for analyzing the data [49 (link)].

The effect of AEA on the membrane potential of MDRSA CI-M was analyzed using the BacLight Membrane Potential Kit (Molecular Probes, Life Technologies) according to the manufacturer's instructions^{63 (link)}. MDRSA was treated with various concentrations of AEA in TSBG for either 2 h at 37 °C or AEA was added immediately before analyzing the membrane potential. The bacteria were resuspended in PBS and 3,3′-diethyloxacarbocyanine iodide (DiOC₂(3)) was added to a final concentration of 30 μM and after 30 min the bacteria were analyzed on flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences) using the 488 nm excitation laser and collecting the data using the green (530 nm) and red (610/620 nm) filters. The BD FACSDiva software was used for the collection of data and the FCS Express 7 software for analyzing the data.

The immediate effect of AEA on the membrane potential of S. mutans was analyzed using the BacLight Membrane Potential Kit (Molecular Probes, Life Technologies, Eugene, OR, USA), according to the manufacturer’s instructions. An overnight culture of S. mutans was resuspended in PBS at an OD_600nm of 0.3 and exposed to various concentrations of AEA. Immediately thereafter, 3,3′-diethyloxacarbocyanine iodide (DiOC2(3)) was added to a final concentration of 30 μM. After 30 min, the bacteria were analyzed by flow cytometry (LSR-Fortessa flow cytometer, BD Biosciences) using the 488 nm excitation laser, and the data were collected using the green (530 nm) and red (610/620 nm) filters [22 (link)]. The BD FACSDiva software was used for the collection of data, and the FCS Express 7 software was used to analyze the data [22 (link)].

The effects of carvacrol on the membrane potential/cytoplasmic membrane depolarization activity were determined according to the BacLight™ Membrane Potential Kit instructions (B34950, Molecular Probes Inc, Eugene, OR, USA).
Sample preparation: Five-milliliter samples of S. pyogenes ATCC 19615 (1 × 10⁸ CFU/mL) were treated with carvacrol (2 × MIC, 1 × MIC, 1/2 × MIC; MIC = 125 µg/mL) or DMSO vehicle control. Ten microliters of 500 µM of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a proton ionophore, were added to 1 mL of each sample. The samples were incubated at 37 °C at 5% CO₂. At 30 min, 1 h, 16 h, and 24 h, the cells were harvested and diluted in BHI to approximately 1 × 10⁶ CFU/mL. The cells (1 mL) were stained with 10 µL of 3 mM of 3, 3′-diethyloxacarbocyanine iodide [DiOC2(3) (Molecular Probes, Eugene, OR, USA)], a membrane potential-sensitive fluorescent probe, for 30 min at 37 °C.
FCM: Flow cytometry was performed at excitation and emission wavelengths of 622 and 670 nm, respectively. Background fluorescence resulting from the medium was determined. Ten thousand events were recorded for each sample. The data were expressed by mean fluorescence intensity (MFI).

Membrane potential was measured using the BacLight Membrane Potential Kit (Molecular Probes) following the manufacturer's recommendations. Membrane potential in this assay is based on the shift between the green fluorescence of DiOC₂ to red in the cytosol of bacteria with higher membrane potential. The proton ionophore CCCP was used as a positive control for disrupting membrane potential. LAC was cultured with 50 nM AIP1 for 5 hr in TSB in the presence of savirin (5 µg ml⁻¹) or vehicle control. After diluting into TSB, the bacteria were incubated with 30 µM DiOC₂ in the dark for 16 min prior to analyzing by flow cytometry (Accuri, C6). Measurements from both the red and green channels were taken and data presented as a ratio of red channel divided by the green channel to reflect the shift to greater change in membrane potential.

For analyzing alterations in membrane potential, control and AA-treated bacteria were resuspended in 1 ml of PBS containing 30 μM of the potentiometric dye DiOC2(3) (BacLight Membrane Potential Kit, Molecular Probes, Life Technologies, Eugene, OR, USA) (Wolfson et al., 2023 (link)). The fluorescence intensities were measured in a Fortezza flow cytometer using the excitation/emission of 488 nm/530 nm for green fluorescence and 488 nm/620 nm for red fluorescence. A relative increase in red fluorescence in comparison to green fluorescence is a sign for membrane hyperpolarization.