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A mechanism of the cytotoxicity of plasma-activated medium (PAM) is revealed by examining the intracellular effects of reactive oxygen nitrogen species (RONS) and lipid oxidation. PAM is cell culture medium activated by irradiation using non-equilibrium atmospheric-pressure plasma using pure Ar gas in ambient air. Extracellular RONS in PAM induced the apoptotic death of HeLa cells. Temporal changes in intracellular RONS, such as ONOO − , NO, and O À 2 , were analyzed. Intracellular RONS generation in HeLa cells following incubation in PAM triggered activation of the caspase cascade pathway and lipid peroxidation of intracellular membranes to induce apoptosis. K E Y W O R D S non-equilibrium atmospheric-pressure plasma, plasma medicine plasma-activated medium (PAM), reactive oxygen and nitrogen species (RONS)

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Octahedral particulates several tens of microns in size were synthesized in a culture medium irradiated through contact with a plume of non-equilibrium atmospheric-pressure plasma (NEAPP). The particulates were identified in the crystalline phase as calcium oxalate dihydrate (COD). The original medium contained constituents such as NaCl, d-glucose, CaCl2, and NaHCO3 but not oxalate or oxalic acid. The oxalate was clearly synthesized and crystallized in the medium as thermodynamically unstable COD crystals after the NEAPP irradiation.

The multiplex coherent anti-Stokes Raman scattering microscopy allowed label-free visualization of cytoplasmic lipid droplets (LDs). The LDs, which act to conserve energy storage, are usually accumulated during the normal apoptosis of HeLa cells with activation of caspase-3/7 leading to downregulation of the fatty acid catabolism pathways. During cultivating in nonthermal plasma-activated medium (PAM), while the activation of caspase-3/7 was induced, the authors found that a dynamic exhaustion of the intracellular LDs, underlying the metabolic mechanism of the PAM-induced apoptotic cell death of HeLa cells.

Oxalate was synthesized in the glucose solution by irradiation with non-equilibrium atmospheric pressure plasma (NEAPP), in which the NEAPP plume contacted the solution surface, via the generation of several intermediate organic products such as gluconic acid. A thermodynamically unstable phase of calcium oxalate dihydrate crystallized rapidly during incubation of a NEAPP-irradiated glucose solution that contained calcium ions and was buffered at neutral pH. Longer irradiation times increased the growth rate and the number of seed crystals.

Interactions between non-equilibrium atmospheric-pressure plasma (NEAPP) and living cells were examined using multiplex coherent anti-Stokes Raman scattering (CARS) microscopy. Our multiplex CARS analyses revealed that NEAPP irradiation generates short-lived radicals that induce a decrease in the mitochondrial activity of budding yeast cells.