Selectfx nuclear labeling kit

Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 30 min. For filipin staining cells on coverslips were stained with 25 μg/ml filipin (Sigma) in PBS in the dark for 2 h at RT, washed with PBS, mounted in Fluoromount G (EMS), and imaged. In some experiments nuclear staining was performed using SYTOX Green or 7-AAD from SelectFX Nuclear Labeling Kit (Invitrogen). For PFO∗ staining cells were permeabilized with 0.1% TX-100 in Cell Staining Buffer (BioLegend) for 30 min and then blocked for 1 h in Cell Staining Buffer. iFluor-647-PFO∗ (Codex Bio solutions and AAT Bioquest, custom order) was then added in Cell Staining Buffer for 1 h. Cells were washed three times with PBS and stained with 1:10,000 HCS CellMask Green (ThermoFisher) and 1:5000 Hoechst 33342 (ThermoFisher) in PBS for 15 min. Cells were washed with PBS and imaged.

For membrane depolarization and permeability assays, bacterial density was adjusted to given values between 5 × 10⁶ and 5 × 10⁷ CFU/ml. Testing solutions and mixtures were prepared as described in section 5.4. Volumes of treatment combinations were 200 μl, experiments were carried out in 96 well microtiter plates in duplicates. Mixtures were incubated in a thermoshaker (Stat Fax® 2200, Awareness Technology, Florida, USA) for 180 min at 27 + − 0.1 °C with continuous shaking at 200 rpm. Fluorescent indicator dyes were then added, and co-incubated for 60 min.
The fluorescent dye DIBAC₄(3) (Biotium Inc., CA, USA) stock solution was 1 mg/ml dissolved in DMSO. This was further diluted 1:1000 in the experimental mixture.
SYTOX® Green and TO-PRO®-3 fluorescent dyes were from the SelectFX Nuclear Labeling Kit (Invitrogen, CA, USA). SYTOX® Green (60 μg/ml in DMSO) and TO-PRO®-3 (210 μg/ml in DMSO) stock solutions were both diluted 1:300 as recommended by the manufacturer.
Fluorescence was then recorded by an iBright™ FL1000 Imaging System (Thermo Fisher Scientific Inc. MA, USA) in the GFP channel. Quantification was performed using the NIH ImageJ [56 ] software ReadPlate2.1 plugin.