Hematoxylin Eosin sections of nude mouse skin after plasma treatment.

<p>Epidermis, dermis, hypodermis and muscle are affected for doses higher than 113 J/cm², 281 J/cm² and 394 J/cm² respectively. In the diagram in the lower quadrant, the doses necessary to produce mild lesions for each layer of the skin are shown.</p

Oxydative stress assay (A-D), Electroporation assay (E-F).

<p>For both tests, nucleuses were labelled with NucBlue, images were taken with the same parameters as for the negative control. Oxydative stress assay: Cells were incubated one hour after treatment by plasma or menadione 100µM with Cellrox deep green, an intracytoplasmic fluorescent marker of oxidative stress. A, negative control, almost no fluorescence is apparent. B, Menadione, a strong cytoplasmic red fluorescence is present. C and D, medium and intense fluorescence depending on the plasma dose, 169 and 508 J/cm² respectively. Electroporation assay: Cells were incubated just before plasma treatment with Sytox green, a fluorescent marker of membrane impairment. E, negative control, almost no fluorescence is visible. F, high-dose plasma treatment (506 J/cm²), a strong green fluorescence is present.</p

Annexin V / PI assay on HMEC and Jurkat cells 8 and 24 Hours after plasma treatment.

<p>Cells were treated for 30 to 120 sec and from 75 to 900 Hz frequency. The administered dose is expressed in J/cm². Flow Cytometry data are analysed on a four quadrant dot plot gated on a region including cells and debris. Propidium iodide (PI) signal is detected in the Phycoerythrine channel, on the Y axis. FITC-labelled Annexin V signal is detected on the X axis. Non-apoptotic, non-necrotic, living cells are located in the lower left quadrant. Results are expressed as median +/- standard deviation. On the right, for each experimental time, a histogram including statistical analyses summarizes cytometry data. Statistical significance: * means p<0.05 when compared with the control; ** means p<0.05 when compared with the immediately lower dose, *** means p<0.05 when compared with the control and the immediately lower dose. On Jurkat cells, 8 hours after treatment, the type of cell death is mainly apoptosis, up to 255 J/cm². Above this dose, the type of cell death is mainly necrosis. At H24, the pattern is non-specific, consistent with late apoptosis or necrosis. Overall the data are consistent with the hypothesis that NPP induces apoptosis on Jurkat cells up to the dose of 255 J/cm². On HMEC, 24 hours after treatment, at 510 J/cm², there is an increase of Annexin V-only positive cells consistent with apoptosis. Otherwise the pattern is mainly necrotic. On HMEC and Jurkat cells, there is a dose effect. To reach a given percentage of dead cells, HMEC cultures need higher doses than Jurkat cells.</p

Electron microscopy of nude mouse epidermis after plasma treatment.

<p>For each sample, on five different fields, 100 cells were counted and classified as normal, necrotic or apoptotic. Results were expressed as median +/- standard error. Representative pictures are shown: A: Normal skin. B: 113 J/cm². Numerous apoptotic keratinocytes are present, presenting highly condensed chromatin. C: 281 J/cm². The epidermis is completely disrupted, no cell is distinguishable. On the control samples, the normal, apoptotic and necrotic cell percentages were 74%±6, 24%±4 and 1% ±0.5 respectively. At the doses of 113 J/cm, the normal, apoptotic and necrotic cell percentages were 20%±4, 68%±6, 12%±3 respectively. These results were statistically different from the results of normal samples (p<0.001). At the dose of 281J/cm² normal cells 0%±0, apoptotic cells 0%±0, necrotic cells 100%±0. These results were statistically different from the results of samples treated at 113J/cm² (p<0.0001).</p