Figure 2. Generation of ROS and RNS by air plasma.

(a) Levels of extracellular H₂O₂ were determined in culture or non-culture (Medium only) supernatants, with (AP) or without (Non-treated) air plasma treatment. The culture supernatant was harvested at the indicated times following air plasma treatment (AP). AP 0 (min) indicates supernatant harvested immediately after plasma treatment. The concentration of H₂O₂ in the culture supernatant was determined by comparison to a H₂O₂ standard calibration curve, following incubation with Amplex UltraRed reagent (n = 5). (b) Generation and/or penetration across the plasma membrane of ROS including H₂O₂, OH⁻, and •O₂ in the intracellular matrix following air plasma jet (AP) were determined using the ROS-sensitive probe H₂DCFDA (n = 5). The fluorescence of untreated cells (Non-treated) was arbitrarily set to 1. (c) Levels of extracellular NO were determined in non-culture (in the absence of HeLa cells, upper) and culture (in the presence of HeLa cells, bottom) supernatants at the indicated times after air plasma jet exposure by the Griess assay (n = 10). (d) Levels of intracellular NO were evaluated using DAF-FM. Data are shown as the mean ± S.E.M. (n = 10).