The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in ...The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in differentiated cells by surface antigen and analysis of cells divisions＇ number by vital dye dilution. Lymphocytes were activated by anti-CD3 Abs with IL-2 presents and grown in vitro for 7 days. Cells division＇s number was measured by dilution of CFSE vital dye which cells were stained previously activation. For telomere length measurement we used flow-FISH method with Cy3 labeled telomere PNH probe. Using this method we evaluated the dynamic of telomere length in CD4＋ and CD8＋ T-cells after 7 days culturing in vitro and revealed the difference in telomere lengthening and shortening versus division rounds in cell subsets. In CD8＋ cells telomeres start lengthen on a second division with the maximum on 4th division round becoming more that 20% longer compared with undividing cells. In CD4＋ cells telomeres did not have any length peculiarities through all division rounds demonstrating different telomere regulation in subsets. This probably occurs due to the higher level ofhTERT protein expression in CD8＋ than CD4＋ cells do.展开更多
文摘The purpose of present work is a measurement of telomere length dynamic in proliferating cells in vitro by modified flow-FISH method. This method is a combination of two modifications： telomere length measurement in differentiated cells by surface antigen and analysis of cells divisions＇ number by vital dye dilution. Lymphocytes were activated by anti-CD3 Abs with IL-2 presents and grown in vitro for 7 days. Cells division＇s number was measured by dilution of CFSE vital dye which cells were stained previously activation. For telomere length measurement we used flow-FISH method with Cy3 labeled telomere PNH probe. Using this method we evaluated the dynamic of telomere length in CD4＋ and CD8＋ T-cells after 7 days culturing in vitro and revealed the difference in telomere lengthening and shortening versus division rounds in cell subsets. In CD8＋ cells telomeres start lengthen on a second division with the maximum on 4th division round becoming more that 20% longer compared with undividing cells. In CD4＋ cells telomeres did not have any length peculiarities through all division rounds demonstrating different telomere regulation in subsets. This probably occurs due to the higher level ofhTERT protein expression in CD8＋ than CD4＋ cells do.
