Anionic Dextran Staining of S. mutans

Overnight cultures of S. mutans were diluted in fresh BHI media to an OD of 0.2. Then, the cells were incubated in the absence or presence of various concentrations of AEA (1.56–50 µg/mL) and 0.05% ethanol for 2 h in fresh BHI media at 37 °C in 95% air/5% CO₂. After incubation, the samples were stained with 10 µg/mL Alexafluor⁶⁴⁷ -conjugated anionic Dextran 10,000 (Invitrogen Live Technologies Corporation, Eugene, OR, USA) for 20 min at 37 °C in 95% air/5% CO₂. Fluorescence intensity was analyzed by flow cytometry (LRS-Fortessa flow cytometer, BD Biosciences, San Jose, CA, USA) using the 640 nm laser excitation, and the fluorescence was collected using the 670 nm filter [13 (link)].