Category — FISH

Baerlocher GM, Vulto I, de Jong G, Lansdorp PM Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3, Canada. . Nat Protoc. 2006;1(5):2365-76.

Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2-3 days.

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Gabriela M. Baerlocher1 and Peter M. Lansdorp. . Cytometry Part A 55A:1-6, 2003.

Telomeres are essential protein-DNA structures at the end of chromosomes which are implicated in genome stability and cell replication. The average length of telomere repeats can be measured by in situ hybridization and flow cytometry [flow-FISH]. Such telomere length values reflect telomere shortening (resulting from cell divisions, oxidative damage and other causes) and telomere elongation (mainly resulting from telomerase activity) of the chromosome-specific telomere length inherited in the gametes. Here we report improvements in flow-FISH methodology that enable measurements of telomere length in subsets of human nucleated blood cells. Methods and Results: In order to measure the telomere length in granulocytes, naive T cells, memory T cells, B cells and natural killer (NK)/NKT cells within a blood sample, we combined flow-FISH with antibody-staining (Multicolor flow-FISH). Most steps in the staining protocol were automated using a 96-well microdispenser device. The minimum detectable difference in telomere length and the reproducibility of the method are in the range of 0.2- 0.5 kb and measurements can be made with as few as a thousand cells.

Conclusions: Automated multicolor flow-FISH will greatly facilitate studies of telomere length regulation in subsets of nucleated blood cells, especially when only few cells are available and when differences in telomere length are small.

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