Dioc2

Bacteria were grown with shaking at 37°C until the exponential growth phase. Cultures were diluted to approximately 1 × 10⁶ CFU/ml in filtered phosphate-buffered saline (PBS), and DiOC₂ (Sigma-Aldrich, Stockholm, Sweden) was added to a final concentration of 30 µM. Depolarized control samples were supplemented with CCCP (Sigma-Aldrich, Stockholm, Sweden) to a final concentration of 1 µM. After 15 min of incubation at 37°C, the florescence intensities of green fluorescent protein (GFP) and Texas Red were measured with a MACSQuant VYB (Miltenyi Biotec, Inc., Bergisch Gladbach, Germany). The ratio of red over green fluorescence was used to determine the membrane potential of each strain (49 (link)).

P-gp, MRP, and BCRP functionality were assessed using rhodamine 123 (Sigma), DiOC2 (3,3′-diethyloxacarbocyanine Iodide) (Sigma), citalopram (Sigma), and doxorubicin (Sigma). Both channels were pretreated with 50 µM verapamil (Sigma), 10 µM MK571 (Sigma), or 1 µM Ko143, which are inhibitors of P-gp, MRPs, or BCRP, respectively. At 30 min after pre-treatment with inhibitors, rhodamine 123 (2 µM), DiOC2 (2 µM), citalopram or doxorubicin (5 µM) in the presence or absence of inhibitor was dosed to brain endothelial channel with flow rate at 100 µl h⁻¹. To monitor the barrier integrity, 100 µg ml⁻¹ of 3 kDa dextran-cascade blue (Thermo Fisher) was dosed simultaneously. We collected apical and basal effluent for 6 h and tracers in the effluents were quantified by measuring fluorescent intensity. The fluorescence was measured at 485/530 nm, 482/497 nm, or 470/585 nm to quantify rhodamine 123, DiOC2, or doxorubicin, respectively, using a Synergy H1 microplate reader (BioTek, USA). Amount of citalopram in the apical and basal media was quantified using mass spectroscopy. The increase of BBB permeability of the drugs in the presence of inhibitor was presented as ‘ratio of P_app’.

The membrane potential (PMF) of AP-R_CLIN-EVO and AP-R_LAB-EVO with or without nitrite was measured as follows: bacteria were cultured with or without 20 mM nitrite and incubated at 37°C with 200 rpm for 6 h. Then, the cells were labeled by 3 mM DiOC₂ (Sigma) in the dark at 30°C for 30 min. The samples were analyze using a BD FACSCalibur flow cytometer with an excitation wavelength of 488 nm and emission wavelength of 610 nm. Gates for bacterial populations were based on the control population by using forward versus side scatter and red versus green emission. The diverse ratios of red and green indicated fluorescence intensity values of the gated populations. Membrane potential was calculated according to the following: log(10^3/2 × [red fluorescence/green fluorescence]). Three repetitions were performed.