Checks had been executed in triplicate and every experiment was independently repeated at least a few times. One-way and two-way analyses of variance ended up performed with Bonferroni correction for numerous comparisons. Statistical importance was decided on at .05 with p<0.05 (), p<0.01 ( ), and p<0.001 (). Statistics and graphics were generated using PRISM GraphPad 5.0. (GraphPad Software, San Diego).As a starting point, 50% hPDL confluent cells maintained in 3% FCS were treated with HeGIW at increasing voltage and exposure time. Twenty-four hours after treatment, we observed a significant decrease of adherent cell number with 5kV and 30 sec exposure, compared to control (Fig 4a and 4b). With these settings, the cell morphology shifted from normal elongated to refringent 6 h after treatment, and most cells floated in the medium culture 24 h after exposure (Fig 4c). During preliminary experiments, we also observed that extracellular environment had a significant impact on cell fate after He-GIW treatment. Indeed, while exposed hPDL cultures maintained in 1% FCS exhibited a dramatic loss of adherent cells compared to control, increasing FCS concentration (Fig 4d) or confluence (Fig 4e) were able to protect cells against loss of adhesion. However, FCS protection was limited and reached a 50% cell recovery plateau starting at 5% FCS concentration (Fig 4d). Based on these findings, we decided to focus next on the mechanisms inducing cell detachment using 5 kv and 30-second exposure length for plasma setting and 3% FCS, 50% confluence for cell culture.Microscopic observation after He-GIW treatment shows that floating cells exhibit a round, balloon-like, morphology without any membrane blebbing (Fig 4c and Fig a in S1 File) suggesting induction of a cell death, mainly by necrosis. To confirm this observation, cells were stained with annexin V, a biochemical marker of apoptosis, and propidium iodide (PI), a marker of cell membrane integrity. Cell treatment with the prototypical apoptosis inducer, staurosporine, showed the appearance at 6 hours of a cell population that was Annexin V+/PI-. In contrast, He-GIW treatment elicited the appearance at the same time point of a cell population that had taken up PI but was heterogeneous with regard to Annexin V staining (Fig 5a and 5b). To evaluate the integrity of Annexin V+ cells under these conditions, we plotted the forward (FSC) and side (SSC) scatter profile of Annexin V+ cells against the total cell population. Unlike Annexin V+ staurosporine-treated cells, which at 6 hours had a scatter profile consistent with intact cells, Annexin V+ He-GIW-treated cells had an FSClow profile consistent with damaged cells (Fig b in S1 File). This, in combination with the fact that they were also PI+ (Fig 5a and 5b), suggests that these cells may11311902 have passively taken up Annexin V due to their damaged state [31]. Later, 24h after treatment with He-GIW or staurosporine, nearly all cells were double positive for Annexin V and PI (Fig 5a and 5b) and damaged (Fig b in S1 File). Although it Fig 4. Device settings 
and cell culture parameters ITE influence cell fate. (a) hPDL were treated with HeGIW using different voltages and (b) exposure lengths.