Triton x 100

Cells grown on glass coverslips were washed with PBS, fixed with 100% ice-cold methanol (AppliChem, Darmstadt, Germany) for 10 min at −20 °C, then permeabilized using 0.1% Triton X-100 (Carl Roth, Karlsruhe, Germany) for 10 min at room temperature (RT). After washing with PBS, cells were incubated in 10% FBS/PBS for 30 min to block unspecific binding. Cells were incubated with IER2 antibody (ARP34401_P050; Aviva Systems Biology, San Diego, CA, USA) diluted 1:500 in blocking solution for 3 h at RT, then extensively washed with PBS. Incubation with the goat anti-rabbit AlexaFluor 546 (Thermo Fisher Scientific, Waltham, MA, USA) secondary antibody (1:1000) was performed for 1 h at RT. Cell nuclei were counterstained with 1 μg/ml DAPI (AppliChem, Darmstadt, Germany) for 5 min at RT, and coverslips were mounted in Fluoromount mounting medium (SouthernBiotech, Birmingham, AL, USA). Fluorescent signals were captured using a Zeiss Axio Imager D1 microscope equipped with an AxioCam MRm camera and AxioVision 4.7 software (Carl Zeiss, Oberkochen, Germany).

The tissue samples were sectioned vertically (thickness: 5 µm) 90° to the gas plasma-treated side (facing up) on Superfrost plus slides (Thermo Fisher, Bremen, Germany) and stored at −80 °C until further staining. Prior to staining, the sections were fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) and washed three times for five minutes in phosphate-buffered saline (PBS). Cell membrane permeabilization was performed for two minutes using 0.25% Triton X-100 (Carl Roth, Karlsruhe, Germany) followed by three washing periods of five minutes in PBS. The TUNEL kit (Roche Diagnostics, Basel, Switzerland) was used for apoptosis detection as per the manufacturer’s instructions. The slides were incubated for one hour at 37 °C in a humidified chamber. The sections were washed three times in PBS followed by DAPI addition to counterstain nuclei. The slides were mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, Taufkirchen, Germany). Imaging and analysis were performed using a high-content imaging device (Operetta CLS; PerkinElmer, Hamburg, Germany) and its associated software (Harmony 4.9; PerkinElmer, Hamburg, Germany). Quantification was based on algorithm-driven object segmentation.

HepG2 (1.5 × 10⁴) and MCF-7 (2.5 × 10⁴) cells were seeded onto glass cover slips in 24-well plates (Sarstedt AG & Co). After 24 h, cells were treated with three different concentrations of CPA, PPA or 4-OH-CPA^UPO at a range of 0–50 µM for HepG2 cells and 0–25 µM for MCF-7 cells in standard medium. 24 h after substance treatment, cells were fixed in 4% paraformaldehyde (Merck Millipore, Massachusetts, USA) for 10 min, washed with PBS and permeabilized in 0.25% Triton X-100 (Carl Roth GmbH) for 3 min. After washing with PBS, cells were incubated for 1 h at RT with anti-phospho-Histone H2A.X (Ser139) mouse monoclonal IgG (clone JBW301, Cat. No. 05–636, Merck Millipore) diluted 1:1000 in PBS containing 1% BSA. After washing, cells were incubated for 1 h at RT with polyclonal Cy3-conjugated goat-anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove) diluted 1:200 in PBS containing 1% BSA and 0.2 µg mL⁻¹ DAPI (Carl Roth GmbH). Immunofluorescence was evaluated with an Olympus IX81 fluorescence microscope (Olympus, Tokyo, Japan).

PMEC were seeded on 12 mm coverslips (Carl Roth) in 24-well plate (Biochrom) at a density of 10 000 cells/well. After two days of culturing, medium was discarded, and coverslips were washed twice with PBS and fixed with ice-cold methanol (−20 °C, Carl Roth) for 20 min. Cells were permeabilized with 0.2% Triton X-100 (Carl Roth), diluted with PBS for 5 min and washed twice with PBS. Non-specific binding sites were blocked by incubating the coverslips with 10% FBS in PBS for 30 min at room temperature. Coverslips were washed twice with PBS and incubated with mouse anti-cytokeratin 18-fluorescein isothiocyanate (anti-Cy18-FITC, Sigma-Aldrich) and mouse anti-alpha-smooth muscle actin antibodies (clone 1A4, Sigma-Aldrich), respectively in a humidified chamber for 1 h. Coverslips were washed three times with PBS. Bound anti-alpha-smooth muscle actin antibody was visualized by 1 h incubation of the coverslips with goat anti-mouse FITC-labeled secondary antibody (Sigma-Aldrich). Nuclei of the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI, Carl Roth) for 15 min. Coverslips were washed twice with PBS, air dried and mounted with 1,4-diazabicyclo[2.2.2]octane (DABCO) on glass slides (both from Carl Roth). Coverslips were analyzed by immunofluorescence microscopy (Microphot-FXA, Nikon, Düsseldorf, Germany).

Microbial strains used as indicator organisms and corresponding incubation conditions are reported in Table 4.
The microbial stock cultures were stored frozen in liquid growth medium at −80 °C with glycerol at a final concentration of 15% (v/v) until required. Spore suspensions of the filamentous fungi were prepared by suspending conidia from 7- to 10-day-old cultures in 10 mL 0.05% (v/v) Triton X-100 (Carl Roth) and were stored at 4 °C [50 (link)]. For recovery of bacterial and yeast strains, one loop from each stock suspension was streaked on appropriate agar plates and incubated for a period sufficient to obtain single colonies. Microbial cells and spores were enumerated with three replicates by a plate-count method for viable cells. Serial dilutions of each cell or spore suspension were plated by surface spreading onto suitable agar medium and the number of visible colonies reported as colony-forming units per milliliter (CFU/mL). Each microbial culture was adjusted to 1 OD₆₀₀ unit with a standard curve relating optical densities at 600 nm (OD₆₀₀) to viable cell numbers [51 (link)]. This allowed the standardisation of assay inoculums by OD₆₀₀ measurements with an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Uppsala, Sweden).

To analyze colony-forming units in the ear, bacteria were isolated from infected ears by homogenization with 15 mL disposable tissue grinders (Fisherbrand) or 15 mL tapered tissue grinders (Wheaton). The cells were centrifuged (16500 g, 3 min, 4 °C) and lysed with 1% triton-X 100 (Roth) in H₂O. The bacteria were serially diluted in triplicates and plated on agar plates.