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8-oxo-dG and its analogs were used as biomarkers of oxidative DNA damage Glafenine and oxidative stress. To evaluate Ag-NP-induced oxidative stress in NSCs, an 8-oxo-dG ELISA assay was utilized as previously described (Liu et al., 2013), according to the manufacturer's instructions (Trevigen, Gaithersburg, MD, USA). Western blot of the pro-apoptotic gene bax Bax expression at the protein level was evaluated using Western blotting procedures as previously described (Wang et al., 2005); Western blots of ��-actin served as an internal control. Statistical analysis Statistical analyses were performed and graphs were produced using GraphPad Prism5. Data are expressed as means �� SDs. Data from the LDH and MTT assays, 8-oxo-dG assay and densitometry value of Bax were analyzed using One-Way ANOVA, with Tukey's post-hoc analysis to compare all pairs of groups. TUNEL and EdU incorporation assays were analyzed using unpaired t-test. All analyses were two-tailed and considered to be statistically different when a p buy Lenvatinib in triplicate and experiments were repeated three times independently. Results On day in vitro (DIV) 2, the seeded NSCs were observed attaching to the bottom of culture dishes/wells. Over the following days, more bipolar NSCs emerged and began forming spheres, indicating their capability to proliferate. The experiments were performed on DIV 8. Both human and rat embryonic NSCs were verified using the NSC markers SOX2 and/or nestin and counterstained with DAPI. As shown in Figures 1A�CF, the immunocyto-chemical stains demonstrated that the majority of the human NSCs were nestin and SOX2-positive. Similarly, most rat NSCs were stained with nestin and SOX2 (Figures 1G�CJ). Figure 1 Representative photographs of immunocytochemical staining of nestin and SOX2 of human and rat NSCs. The evenly distributed neural stem cells (human) were positively stained with nestin (A, green; fluorescence) and SOX2 (D, Green; fluorescence), indicating ... Ag-NP-induced cytotoxicity in NSCs LDH is a cytoplasmic find protocol enzyme sequestered inside viable cells with intact plasma membranes. It is released from cells with damaged membranes, thus, the amount of LDH released from cells into the culture medium is taken to indicate some level of toxicity. As shown in Figures 2A,B, no significant effect on extracellular LDH concentration was detected at the lowest dose of 1 ��g/ml. At doses of 5 ��g/ml and higher, however, Ag-NP exposure resulted in significant, dose-related increase in LDH release from both human [F(4, 24) = 69.70, p