Electrical high voltage and current signals analyzed during one impulse (2a) and during one period (2b) (He gas flow = 2slm, voltage frequency = 10kHz, voltage amplitude = 5kV, voltage duty cycle = 1).

<p>Electrical high voltage and current signals analyzed during one impulse (2a) and during one period (2b) (He gas flow = 2slm, voltage frequency = 10kHz, voltage amplitude = 5kV, voltage duty cycle = 1).</p

Main emissive species produced with Helium as plasma carrier gas.

<p>Main emissive species produced with Helium as plasma carrier gas.</p

Mitochondrial membrane depolarization precedes cell membrane alteration.

<p><b>(a)</b> Adherent cell percentage was monitored for 6 h after He-GIW treatment. <b>(b)</b> Cell Δψm was monitored by FACS analysis for 6h using DiOC6 staining. Results of one representative experiment of at least three. <b>(c)</b> Geometric means of DiOC6 staining relative to control 1, 3 or 6 h after He-GIW treatment. <b>(d)</b> Δψm (DiOC6) and plasma cell membrane permeability (7AAD) were monitored by FACS analysis for 6 h after He-GIW treatment. When present, error bars represent S.E.M. of three independent experiments p<0.05 (*), p<0.01 (**) and p<0.001 (***)</p

Experimental set-up.

<p>Experimental set-up.</p

Relative concentration of the main emissive species in the guided ionization wave (conditions: He gas flow = 2slm; voltage frequency = 10kH; voltage amplitude = 5kV; voltage duty cycle = 1%.

<p>Optical fiber placed 5mm after the exit ceramic tube).</p

Device settings and cell culture parameters influence cell fate.

<p><b>(a)</b> hPDL were treated with He-GIW using different voltages and <b>(b)</b> exposure lengths. Adherent cell number was evaluated 24h after treatment and expressed as a percentage of control. <b>(c)</b> Cell morphology was monitored by phase contrast microscopy 6h and 24h after treatment. <b>(d)</b> Cell culture incidence on He-GIW treatment was evaluated using different FCS concentrations and <b>(e)</b> cell confluence. Adherent cell number was assayed 24h after treatment and expressed as percentage of control. Error bars represent S.E.M. of three independent experiments p<0.05 (*), p<0.01 (**) and p<0.001 (***).</p

He-GIW induces a necrotic cell death.

<p><b>(a-c)</b> Presence of apoptosis was assayed by FACS analysis of Annexin V/PI dual staining. <b>(a)</b> An example of flow cytometry profile 6 h after treatment with He-GIW or with staurosporine, a positive control for apoptosis. X axis represents annexin-V-APC staining and Y axis PI staining. <b>(b)</b> Percentage of cells positive for Annexin V only (black), PI only (white) or both (grey) was measured 6 h and 24 h after treatment with He-GIW or staurosporine in standard cell culture conditions (c) or with 1% or 5% FCS. <b>(a-c)</b> are representative of 3 independent experiments. <b>(d)</b> Caspase 3 cleavage (red) was assayed by immunofluorescence 6h after He-GIW or staurosporine treatment. Cell nuclei are labeled with Hoechst and appear blue. <b>(e)</b> Caspase 3 cleavage was monitored by Western Blot for 2 h after He-GIW treatment. <b>(f)</b> Adherent cell percentage 6 h after He-GIW treatment in presence or absence of caspase inhibitor Z-VAD. Error bars represent S.E.M. of three independent experiments. p<0.05 (*), p<0.01 (**) and p<0.001 (***)</p

Effect of Plasma Activated Liquid (PAL) on bacteria viability.

<p>A: 1: control bacteria incubated for 2 hours in PBS and % of surviving cells was evaluated by the CFU method. 2: <i>E</i>. <i>coli</i> exposed to He plasma for 10 min with 2 hr storage at 4°C. 3: Bacteria exposed to PAL (PBS treated for 10 min He plasma) for 2 hr at 4°C. 4: Bacteria exposed to He plasma for 10 min and incubated for 2 hours with non-plasma treated PBS at 4°C. 5: Bacteria exposed to plasma for 10 min and then incubated for 2 hours at 4°C with PAL (PBS treated for 10 min He plasma). 6: Bacteria exposed to PAL (PBS treated for 10 min He plasma and left at room temperature for 2 hours) for 2 hours at 4°C. The values are means ± SEM of 3 separate experiments (*p<0.01 and ** p<0.05 vs control). B: Same experimental procedures using He-N₂ plasma. C: Same experimental procedures using He-O₂ plasma.</p

Detection of oxidatively modified proteins following plasma exposure.

<p>A: Bacteria exposed to plasma treatment (He, He-O₂ and He-N₂) for 10 min with 2 hours post-treatment storage. To detect oxidatively, modified protein bacterial extracts were treated with 2,4-dinitorphenylhydrazine to derivatize protein carbonyls and then evaluated by SDS-gel electrophoresis using 2,4-dinitrophenyl antibodies. B: Detection of 4-hydroxy-2-nonenal protein modification by ELISA. The values are means ± SEM of 3 separate experiments. C: Western blot analysis using nitrotyrosine specific antibodies. D: Bacterial extracts collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with a dot calibration curve and relative quantity of bacteria lipid A was estimated. The relative intensity of each spot was quantified (Image J).</p

Effect of Plasma Activated Liquid (PAL) on bacteria viability.

<p>A: 1: control bacteria incubated for 2 hours in PBS and % of surviving cells was evaluated by the CFU method. 2: <i>E</i>. <i>coli</i> exposed to He plasma for 10 min with 2 hr storage at 4°C. 3: Bacteria exposed to PAL (PBS treated for 10 min He plasma) for 2 hr at 4°C. 4: Bacteria exposed to He plasma for 10 min and incubated for 2 hours with non-plasma treated PBS at 4°C. 5: Bacteria exposed to plasma for 10 min and then incubated for 2 hours at 4°C with PAL (PBS treated for 10 min He plasma). 6: Bacteria exposed to PAL (PBS treated for 10 min He plasma and left at room temperature for 2 hours) for 2 hours at 4°C. The values are means ± SEM of 3 separate experiments (*p<0.01 and ** p<0.05 vs control). B: Same experimental procedures using He-N₂ plasma. C: Same experimental procedures using He-O₂ plasma.</p