Figure 13

Plasma activated medium (PAM) triggers processes #1 and #2, but not #3. Medium was treated with CAP for 1 min and then an equal volume of cells of double standard density was added. Where indicated, inhibitors were added during the initial contact of PAM and cells. After 25 min of incubation, assays were subjected to three cycles of washing and the cells were resuspended in fresh medium. Where indicated, inhibitors were added at this step. Inhibitors were added at the following concentrations: AEBSF (100 µM); FeTPPS (25 µM), HIS (2 mM). The assays were further incubated and the percentages of apoptotic cells were determined kinetically. Time zero in all assays is the time of mixing PAM and cells. Control assays without PAM did not show apoptosis induction above 4% at all time points (data not shown). The results show that PAM induces the kinetic processes #1 and #2 with equal efficiency and characteristics as shown for direct CAP treatment in the preceding figures, whereas PAM has no potential to initiate process #3. This finding is in line with the short-lived nature of singlet oxygen derived from the gaseous phase of CAP that is responsible for process #3. These findings demonstrate the generation of primary ¹O₂ through the interaction of the long-lived species in PAM and to the role of secondary ¹O₂ that is generated by the tumor cells, dependent on their active NOX1.