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At last 200?��L cold solution C were added and incubated for 10?min in the dark on ice. The cells were filtered through a 40?��m mesh, and analyzed using BD FACS Aria. A total of 10?000 events were acquired. What percentage of the cell population in each part of the cell cycle were analyzed based on the amount of DNA present using the Modfit software. For Oct4, Sox2 and Nanog staining, cells or cytoplasts on glass slides were fixed in 4% paraformaldehyde for 20?min at RT and washed twice with ice-cold phosphate-buffered saline (PBS). The cells were incubated in 50?��L anti-Oct4 mouse anti-human antibody (1:100; Santa Cruz) or anti-Sox2 goat polyclonal IgG (1:50; Santa Cruz) and anti-Nanog goat anti-human antibody (1:25; R&D) or anti-Nanog antibody (1:500; Abcam) diluted in PBS/4% goat or donkey serum overnight at 4��C, washed three times with PBS. Localization Dipivefrine of antigens was visualized by using fluorescein isothiocyanate www.selleckchem.com/products/forskolin.html (FITC) conjugated goat anti-mouse IgG (1:400; Sigma), Alexa Fluor 594 goat anti-rabbit IgG (1:1000; Invitrogen) or Alexa Fluor 594 donkey anti-goat IgG (1:1000; Invitrogen). Slides were washed three times in PBS and mounted with 4?6?-diamidino-2-phenylindole dihydrochloride (DAPI) for analysis by fluorescence microscopy. The cells in which chromosomes were most highly condensed and scrunched up and aligned at the metaphase plate were considered as being at metaphase. The cells in which the sister chromatids separated were considered as being at anaphase. The cells in which the nuclear membrane was intact, the chromatin had not yet condensed and chromosomes were not visible were considered as being at interphase. selleck chemicals In order to estimate the percentage of positive cells, photographs were taken of three independent cultures and of 20 random views of the cells. Two sample t test was used to evaluate statistical significance. P values were considered to be statistically significant when