By Trygve O. Tollefsbol
This ebook investigates a number of the techniques which are plagued by the age of an organism. numerous new instruments for the research of organic getting older were brought lately, and this quantity presents tools and protocols for those new options as well as its assurance of validated strategies. Researchers looking new expertise and methods will locate this quantity of great profit as they stream in the direction of new instructions.
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Extra info for Biological Aging: Methods and Protocols (Methods in Molecular Biology Vol 371)
And Scharffetter-Koehanek, K. (2002) Psoralen plus UVA (PUVA) induced premature senescence as a model for stress-induced premature senescence. Exp. Gerontol. 37, 1197–1201. 02_Tollefsbol 1/25/07 12:57 PM Page 19 Aging Cell Culture Methods 19 19. , and Di Leonardo, A. (2000) Differential gene expression in p53-mediated G(1) arrest of human fibroblasts after gamma-irradiation or N-phosphoacetyl-L-aspartate treatment. Carcinogenesis 21, 2203–2210. 20. Hadley, E. , Kruss, E. , and Cristofalo, V. J.
However, establishing this direct relationship is not a requirement for utilizing cell culture as a tool for studying aging mechanisms. In fact, fibroblasts in culture express human genetic, metabolic, and regulatory behavior allowing the cells to undergo changes in a predictable and reproducibly constant environment (9). 2. Extra-Culture Conditions Cells maintained in culture are continuously exposed to full-spectrum fluorescent light, which can be detrimental to stable cell conditions. This light can affect culture conditions as well as the cells themselves.
After incubation, cells are centrifuged (400g, 5 min) and supernatant discarded. 4. The pellet is resuspended in 3 mL of serum-free DMEM and is immediately used for analysis and sorting (Note 12). 5. ). Sorting gates are defined in Fl1 (logarithmic), conveniently in a FSC/FL1 dotplot. Use the left and right quartiles with respect to FL1 to sort cells with highest (high FL1) and lowest (low FL1) peroxide levels. 2. JC-1 STAINING FOR DETERMINING MMP (NOTE 13) 1. 1 × 105 cells are resuspended in 500 μL of RPMI 1640 phenol-free without supplements (Sigma) and 1 μL of JC-1 stock solution is added to give a final concentration of 1 μg/mL.