Method of Three-Dimensional (3D) Analysis of Telomeric Changes in Primary Mouse and Human Cells
Primary mouse fibroblasts used in our study were isolated from the spleen of normal Balb/c Rb 6.15 and T38HxBalb/c mice. The spleens were removed from the mice; using a scalpel, they were cut into tiny pieces in the presence of a small amount of DMEM/F12 media; seeded onto slides and placed in tissue culture plates for overnight incubation. Next day, 10ml of the same media was added to the plates in the incubators. Media was changed every other day until the cells on the slides were near confluent. The human fibroblasts were obtained from biopsies from Children’s Hospital in Basel, Switzerland. Their diploid karyotype was confirmed by the diagnostic lab of the hospital. Like the mouse fibroblasts, they were grown on slides in tissue culture plates and the media was changed regularly until they were near confluent. 3D telomere analysis was performed on these cells in order to compare their characteristic organization. The cells were 3D fixed on slides using a 3.7% formaldehyde solution and FISH (fluorescence in situ hybridization) was performed using PNA Cy3 telomere probe from DAKO.
Image acquisition was done using a 63x 1.4 oil objective on an Axiophot 2 microscope with exposures of 300 ms for Cy3 in mouse and 1500-1800 ms for Cy3 in the human cells. Images were captures from 80 z-stacks at 0.200μm at a resolution of xy:107nm and z:200nm for each stack. The same conditions were used for both mouse and human fibroblasts. The Axiovision 3.1 software and constrained iterative algorithm (1) were used for deconvolution of the 30 nuclei on each slide. TeloviewTM software 2.0 (2) was used to measure telomere length.
Using this method we could investigate the status of telomere length and aggregate formations at different passages in the human and mouse system. Our preliminary findings show that telomere shortening and telomere aggregates are present in early passages.
References:1.Schaefer et al,J.Microsc.204(Pt 2):99-107,2001; 2.Vermolen et al,Cytometry A.144-50,2005