To determine the possible role of TNF- in controlling cell viability, we investigated U251MG cell viability in the presence of lenalidomide, a TNF- inhibitor. CD86 receptor (M1 marker) expression in plasma-exposed THP-1 cells than in the native cells. In contrast, low CD163 receptor expression levels were detected in THP-1 cells after plasma treatment as confirmed by immunofluorescence (Figure 2G). From these data, we conclude that plasma exposure increased M1-positive PTC299 population in PMA treated THP-1 cells than only PMA-treated cells. Another feature of macrophage differentiation is increased number of certain membrane-bound organelles [23,24]. To confirm the increased numbers of cellular organelles in the cytoplasm, we next stained plasma treated PMA-activated THP-1 cells for mitochondria and lysosomes, whose cytoplasmic number contributes to the differentiation and accumulation of macrophages after the stimulus . Flow cytometer analysis revealed that the plasma-treated THP-1 cells had greater intensity of mitochondrial and lysosomal staining than the native groups (Figure 2H,I). These observations clearly PTC299 suggested that plasma exposure effectively induced macrophage polarization/differentiation in THP-1 cells. 2.3. M1-Like Macrophages Induce Solid Tumor Cell Death If Activated by Plasma with PMA Next, we investigated the possible contribution of plasma-activated macrophages towards anti-cancer activity. Prior to these experiments, we confirmed that plasma treatment did not induce significant cell death in monocytic cells using propidium iodide (PI) staining (Figure 2J). On the other hand, MTT assays showed that a single plasma exposure had the least effect on cell death in U251MG and U87MG solid cancer cells (Figure 3A,B). Given that KIFC1 ATP is the central energy source of PTC299 cells and, therefore, a measure of cellular metabolism and viability, we have further investigated PTC299 the cellular ATP content in glioma cells using cell-titer Glo reagent. The ATP levels of glioma cells were differentially affected by plasma treatment alone in both types of glioma cells, as seen in Figure 3C. Thus, the differential affected ATP levels could be explained by a change in viability induced by plasma treatment in PMA treated THP-1 cells. To observe direct evidence of plasma-stimulated macrophages, we co-cultured these plasma stimulated macrophages with GBM cells, as depicted in Figure 3D. As plasma has been widely shown to induce cell death through ROS [18,26]. We first detected intracellular ROS levels in glioma cells in co-culture condition with plasma stimulated macrophages. The plasma-treated groups had higher levels of ROS in glioma cells than the control groups (Figure 3E). Only PMA-treated THP-1 cells were also able to induce PTC299 ROS in glioma cells in co-culture conditions; however, this effect was boosted by plasma treatment at 1- and 3-min exposure. After two days, the number of viable tumor cells was measured by MTT assays in the same co-culture condition. Plasma-activated macrophages directly affected the cell viability and ATP content of U251MG and U87MG cells compared with those observed in the co-culture condition with supernatant medium (Figure 3F,G). Moreover, caspase-3/7 activation (an indicator for apoptotic cell death) was also increased by the direct co-culture condition in glioma cells (Figure 3H). The growth inhibitory effect of the activated macrophages on glioma cells was also examined by screening the anti-apoptotic gene levels. There was a significant induction of BCL-Xs, ATM, BAX, cleaved caspase-3 and p53 expression in U251MG cells when co-cultured with plasma-stimulated macrophages as seen by western blotting (Figure 3I). Consistently, mRNA levels of p53, CAS3 and BAX were also upregulated in glioma cells when co-cultured with plasma stimulated macrophages in similar conditions (Figure 3J). In addition, the histone 2A family member X (-H2AX) is known to be phosphorylated at serine 139 and forms discrete foci at the DSB sites in response to DNA double-stranded breaks (DSBs) during apoptotic cell death . Accordingly, we next stained glioma cells with -H2AX dye after co-culture with the macrophages and found that the amount of DSBs was highly increased in glioma cells (Figure 3K). Notably, cleaved Poly(ADP-ribose).