As expected, we found that this script is a reliable tool to assess changes in SG formation

luate differences. Results Determining the Appropriate NT-Plasma Dose plasma treatment, although 20142041 the 50 Hz and 3500 Hz treatments were significantly above control. H2O2 induced a slightly higher 5.6% increase in cell death. Results for the 3500 Hz treatment, were an under estimate of cell damage, as large areas of the culture plate were devoid of cells immediately after treatment. Due to cell detachment this setting was not used in subsequent assays. To further assess the 50 and 1000 Hz dose rates, MLO-A5 cell cytotoxicity was measured using lactate dehydrogenase . At 24 h LDH release in response to 50 Hz was 5.1% and 1000 Hz was 7.7% above NTplasma sham control. Both treatments were less than H2O2, which induced an 11.2% increase in cell death. To prevent changes in intracellular oxidant levels, TEMPOL a membrane permeable scavenger of both ROS and RNS, was added 1 hr before NT-plasma or H2O2 treatment. TEMPOL reduced the release of LDH in response to 1000 22576162 Hz NT-plasma and significantly decreased LDH release in response to H2O2, indicating ROS/RNS are being generated in response to NT-plasma and cause cell damage. To determine whether NT-plasma Piceatannol price treatment affected cell proliferation or caused DNA damage, flow cytometric analysis of cell cycle phase was determined 24 hours after treatment of MLO-A5 cells with 50 and 1000 Hz NT-plasma or H2O2. No significant differences were observed in the G1, S or G2/M phase after any treatment, indicating no increase in cell proliferation or blockage of the cell cycle due to DNA damage. Additionally, Western blot analysis was performed to assess damage to DNA or mitochondrial membranes using antibodies to the histone2A variant and cytoplasmic cytochrome c, respectively. The 1000 Hz NT-plasma treatment dose was achieved by varying the time to include 10 sec / NT-Plasma Enhances Skeletal Cell Growth Production of Intracellular ROS in Response to NT-Plasma To evaluate intracellular reactive species levels in response to NT-plasma at 1000 Hz, 10 sec, mitochondrial superoxide anion and cytosolic/mitochondrial H2O2 levels were measured using fluorescent indicators MitoSox and dihydrorhodamine, respectively. Measurements were done immediately posttreatment, at 1 hr and 24 hr after NT-plasma and results were compared to sham controls. Immediately post-treatment and at 1 hr, NT-plasma treated cells showed significantly increased intracellular O22. and H2O2 generation as compared to PRE-PL. Levels of both oxidants returned to PRE-PL levels by 24 hr. The immediate increase in H2O2 may be due to ROS produced by NT-plasma but the extended detection indicates ROS are being actively produced in response to NT-plasma. To confirm the production of ROS in response to NT-plasma treatment, MLO-A5 cells were pretreated for 1 hr with either NAC, to maintain intracellular redox balance and interfere with ROS production or TEMPOL, a scavenger of ROS as well as RNS. After pre-incubation with the inhibitor, the cells were subjected to NT-plasma treatment at 1000 Hz for 10 sec. NAC significantly inhibited the POST-PL and 1 hr NT-Plasma increase in both O22. and H2O2. TEMPOL also significantly inhibited the POST-PL and 1 hr increase in O22., and it inhibited the POST-PL increase in H2O2. To lesser extent TEMPOL inhibited the 1 hr increase in H2O2 in response to NT-Plasma, and at 24 hr H2O2 production was increased. One possible explanation for the decreased effectiveness of TEMPOL after 1 hr and 24 hr after NT-plasma/TEMPOL treat