Fig 1.
Flowing afterglow set up.
Fig 2.
Effect of nitrogen afterglow exposure on bacteria inactivation and viability.
A: Survival curves obtained for E. coli bacteria exposed to the nitrogen late afterglow with and without the MgF2 filter. B: MTT test in bacteria control (vacuum-treated) vs bacteria exposed to the nitrogen late afterglow. Bacteria were eluted from the slides with 1 ml of Broth medium, and incubated in this medium with the MTT reagent (5 mg/ml). After 3 h incubation at 37°C, the bacteria suspension was centrifuged, and the violet formazan crystals were dissolved in 200 μl of DMSO. The optical density was estimated on a microplaque TECAN reader. C. Pictures of bacteria submitted either to vacuum alone for 15 min (LP), or to vacuum + nitrogen late afterglow (AG), and stained with the nucleic acid fluorescent probe DAPI. The results are expressed as % of the vacuum-treated control. Mean +/-SEM of 5 separate experiments, * < p.0.05.
Fig 3.
Effect of nitrogen afterglow exposure on bacteria morphology.
A: Control (E. coli). B, C, D, E: After respectively 5, 10, 20 and 40 minutes of exposure to the pure N2 afterglow.
Fig 4.
Effect of nitrogen afterglow exposure on lipid A.
A. Dot blot binding assay: Increasing concentrations of lipid A were spotted on nitrocellulose membranes and blotted with an anti lipid A antibody. The relative intensity of each spot was quantified (Image J), allowing to build a dose-response calibration curve.B. Dot blots of lipid A pure (left panel) and present in E. coli extracts (right picture): 1 μg pure lipid A was spread off on sterile glass slides, and exposed to vacuum (control), or vacuum + nitrogen afterglow, in the conditions described in the Method section. At the end, the lipid A was eluted, spot on nitrocellulose membrane and immunoblotted with an anti lipid A antibody. The results are expressed as % of residual lipid A vs the vacuum-treated control. On the right panel, determination of the lipid A content in exposed bacteria. 10 μl of a bacterial solution (108/ml), were spotted on glass slides and were treated with plasma. Bacteria extracts were collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with the dot calibration curve and relative quantity of bacteria lipid A estimated. In insert, pictures of lipid A dot-blots pure (left) or from bacteria (right), in vacuum-treated and vacuum + nitrogen afterglow treated conditions. Mean +/-SEM of 5 separate experiments, * < p.0.05.
Fig 5.
Nuclear translocation of the NFκB transcription factor.
On the left, effect of pure lipid A: CRL2181 murine endothelial cells were treated for 20 min with lipid A (200 ng/ml) after low pressure treatment (vacuum), plasma treatment (5 Torr, 200 W, 40 min) (plasma) or from stock solution (untreated). A negative control without lipid A treatment was done (vehicle). At the end, cells were washed, the nuclei were extracted and used for SDS-PAGE electrophoresis and immunoblotting, using an anti NFκB antibody.On the right, effect of lipid A from bacteria: CRL2181 were stimulated for 2 h with 10 μl of E. coli extracts obtained after treatment with low pressure (5 Torr) for 15 min and N2 post-discharge at 200 W for 40 min, or low pressure only, for bacteria control. The nuclei were extracted and used for immunoblotting of NFκB as for pure lipid A.