Falcon round bottom tubes

Cells were disassociated with Accutase™ (Gibco, #A1110501) and pelleted at 200 g for 5 min. Pellet was resuspended with 1 mL of 1X PBS with 10% FBS and filtered through a 35 µm nylon mesh cell strainer cap into a 5 mL Falcon® Round-Bottom Tubes (Corning, #352235). Cells were loaded into the BD FACS Aria Fusion flow cytometer coupled with a BD FACSDiva Software (version 9.0.1). After excluding doublets or non-viable cells, GFP+ events were collected into a tube containing 0.5 ml of cell specific media.

Membrane integrity was assessed by using a propidium iodide (PI) uptake assay, as previously described [41 (link)]. In brief, 2 μL of stationary enterococcal cultures were transferred to Falcon round-bottom tubes (14 mL; Corning), each containing 2 mL fresh BHI medium. Cultures were grown to an OD₆₀₀ of 0.2 at 37 °C with shaking (180 rpm). At OD₆₀₀ of 0.2, compound 2 (at MBC), 5 (at MBC), 10 (at MBC) or nisin (25.6 μg/mL; 1× MIC) was added to the tubes and incubated for 5 min. Then, 15 μL of culture was transferred into 980 μL filtered phosphate-buffered saline, and then stained for 5 min with 5 μL of 0.1 mg/mL PI. After staining, the red fluorescence levels (FL3 channel) in cells were recorded by using a BD Biosciences Accuri C6 flow cytometer (Becton Dickinson). The settings of the flow cytometer were as follows: 25,000 recorded events at an FSC threshold of 15,000, and medium flow rate. The gating of the stained cell population and analysis of flow cytometry data were performed in CFlow (BD Accuri). The assessment of membrane integrity was performed in three biological replicates for each condition.