Even if the authors did not investigate intracellular signaling, it seems that their plasma jet did not generate reactive diffusible ROS. Mitochondria, peroxisome, cytoplasmic proteins and lipid layers constitute the main sources of ROS in eukaryotic cells. Our findings demonstrated that 2 min of treatment with helium generated plasma does not alter the mitochondrial membrane potential in fibroblasts thus excluding these organelles as source of ROS. Administration of cytoplasmic free radical scavengers, on the other hand, significantly dampened the plasmainduced generation of ROS suggesting that cytoplasmic proteins and membrane lipids are the eligible substrate for ROS formation after plasma exposure. Indeed, plasma treatment induced early transient lipid peroxidation in the cytoplasmic compartment. The rapid drop in plasma-induced ROS generation prompted us to hypothesize that just as other human primary cells, HSCs and ISEMFs are endowed with an efficient armamentarium of scavenger enzymes to contain the WY 14643 msds oxidative burst and to avoid mitochondrial involvement thus preserving ATP production. ROS kinetics elicited in primary cells by plasma treatment significantly differed from those described in tumor cells where hypoxia and nutrient deprivation usually result in mitochondrial dysfunctions boosting ROS production and accounting for an increased rate of death of tumor cells in response to oxidative stress. Our results have clearly demonstrated that helium generated plasma treatment induces proliferation and migration of human fibroblast-like primary cells mainly through intracellular ROS formation. Since the threshold of ROS is crucial in cellular signaling and their levels could be modulated in the intracellular compartment by non-thermal atmospheric pressure plasma, it represents a promising tool in the control of fibroblast activation. As outlined in Figure 2B, differences of only fractions of a millimeter in the distance between the plasma needle and the cellular medium affect ROS generation. Moreover, as recently reported, the composition of the medium embedding the samples greatly influences ROS generation in plasma treated cells. Taken together, these data demonstrate that the effects of plasma can be modulated depending on the nature of the sample and the aim of the treatment. At the same, however, it is by now unquestionable that in vivo plasma treatment requires availability of accurate standardized devices as well as in depth knowledge about the biological significance of ROS formation in different cell populations.