S by on a regular basis passage. The shape and growth of iPS cell colonies have been not clearly changed by adding either proprietary antioxidant supplement from SigmaAldrich (AOS) or homemade antioxidant cocktail (AOH) at relative low concentrations in culture medium for two months of followup. Immunostaining showed that all of these iPS cell colonies clearly expressed Oct3/4, Nanog, SSEA4, and ALPSCIENTIFIC REPORTS | four : 3779 | DOI: 10.1038/srep03779www.nature.com/scientificreportsFigure 1 | Stemness of iPS cells immediately after two months of culture. The expression of stem cell markers Oct3/4, Nanog, SSEA4, and ALP have been detected by staining, and representative pictures of expressions in 201B7 (A) and 253G1 (B) iPS cell lines have been shown. Western blot evaluation on the expressions of Nanog and Oct3/4 in 201B7 (C) and 253G1 (D) iPS cell lines was also accomplished, and representative photos that cropped from fulllength blots (Supplementary Figure 1) was shown. Abbreviations: AOS, proprietary antioxidant supplement from SigmaAldrich; AOH, Homemade antioxidant cocktail.after 2 months (Figure 1A and B), indicating that all culture situations maintained “stemness” of iPS cells quite properly. Western blot evaluation also showed that the expressions of Nanog and Oct3/ four at comparable higher levels in all iPS cells below diverse culture situations (Figure 1C and D), while the expressions were not very carefully quantified. Low dose antioxidants decreased the intracellular ROS levels in iPS cells. We 1st measured ROS level by detecting the fluorescence intensity below microscope (Figure 2A). When compared with the control, the addition of proprietary antioxidant supplement from SigmaAldrich or homemade antioxidant cocktail at relative low concentrations in culture medium of course decreased the levels of intracellular ROS inside the iPS cells (upper photos in Figure 2A). Semiquantitative analysis showed that the relative fluorescence intensity of intracellular ROS had been significantly lower within the iPS cells cultured using the addition of antioxidants in medium than that of your handle (decrease bar graphs in Figure 2A).1310405-06-1 Chemscene To further quantitative measure the ROS levels, we measured the fluorescence intensity in iPS cells by flow cytometry (Figure 2B).Price of Gemfibrozil 1-O-β-glucuronide Again, the addition of antioxidants in medium showed to drastically decrease the ROS levels inside the iPS cells, though the lower of ROS by antioxidants was not clearly shown in a dosedependent manner.PMID:23891445 Low dose antioxidants did not market DNA damage or inhibit DNA repair in iPS cells. We evaluated the DNA damage by counting the formation of 53BP1 foci within the nuclei of iPS cells following 2 months culture with all the addition of antioxidants in medium or without the need of. A quantitative evaluation showed that the percentages of iPS cells with 53BP1 foci (Figure 3A,B) within the nuclei, plus the expressions ofSCIENTIFIC REPORTS | four : 3779 | DOI: 10.1038/srepphosphorylated ATM measured by Western blotting (Figure 3C,D) have been not notably diverse among culture conditions. Genomic aberrations in iPS cells soon after 2 months culture. To facilitate direct comparisons, exactly the same iPS cells that had been expanded from a single colony had been utilized to initiate cultures under distinct circumstances in parallel. The information in the array CGH showed some amplifications (red dots) and a few of deletions (green dots), with log2 ratios over 0.75 (Figure 4A, Supplementary Table 1). Compared with all the handle group which was not added antioxidants in medium, the events of genomic aberrations inside the 20.
