Safranin

Mitochondrial membrane potential was assessed as described (Chowdhury et al., 2015 (link)). In brief, Oxygraph-2k was used with O2k-Fluorescence LED2 module equipped with filter sets for safranin (excitation at 495 nm and emission at 587 nm). safranin (Sigma-Aldrich, S2255) dissolved in distilled water up to a final concentration of 2.5 μM. Isolated skeletal muscle mitochondria (65 μg/ml) were suspended in the mitochondrial respiration buffer (see “Measurement of H₂O₂ production”) including 2.5 μM safranin. The membrane potential was tracked live with the addition of ADP (2.5 mM), pyruvate (5 mM), glutamate (10 mM) and malate (5 mM) or ADP (2.5 mM), succinate (10 mM) and rotenone (0.5 μM). In a separate experiment, a standard curve was obtained by adding known amounts of safranin to the assay medium in the presence of mitochondria. The signal was then normalized (0–1) to detect changes after addition of substrates (2.5 μM safranin set as 1).

Amikacin disulfate salt (Acros Organics) as well as, thymol (purity ≥98.5%), Safranin, p-iodonitrotetrazolium (INT) chloride, and Safranin were purchased from Sigma-Aldrich. PLGA (RESOMER RG 503 H, 50 : 50, Mw 24000–38000) was obtained from Boehringer Ingelheim Pharma GmbH&Co. Polyvinyl alcohol (PVAL) was obtained from Roche Diagnostics.

TSB was pretreated with e-cigarette aerosols (10-s puff, 5-min exposure), and overnight cultures were diluted 1:10 in pretreated medium and incubated for 24 h in a 96-well plate (100 μL per well). After 24 h of incubation, plates were washed with deionized water and air dried at 37°C for 20 min. To stain the biofilm, safranin (65092B-95; Millipore Sigma, Darmstadt, Germany; 125 μL containing 6.04g/L safranin, 19% ethanol, and 1% methanol, diluted in water) was added to the wells and incubated at room temperature for 20 min. safranin was discarded, and the wells were washed with deionized water and air dried at 37°C. The biofilm was dissociated by resuspending in ethanol-acetone mix (80:20). The absorbance was read at OD₄₉₀. Results were normalized to blanks, graphed, and analyzed on GraphPad Prism (Prism 8).