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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This work was supported by a US National Institutes of Health grant, the Swiss National Science Foundation, the Bernese Cancer League, Switzerland, and the National Cancer Institute of Canada with funds from the Terry Fox Run. We thank Dirk Roos (Sanquin Research at CLB, Amsterdam, The Netherlands) for advice on the ammonium chloride lysis of red blood cells, and all current and past members of the Lansdorp laboratory for their various contributions to the development of the flow FISH protocol.
G.J. and P.L. are founding shareholders in Repeat Diagnostic Inc., a company specialized in leukocyte telomere length measurements using flow FISH.
Calibration for linearity of the flow cytometer in the fluorescence 1 channel (Fl1) using premixed MESF fluorescence beads. (PDF 4349 kb)
Example of a flow FISH data acquisition template showing human nucleated blood cells hybridized in the presence of fluorescein-labelled PNA that were stained with antibodies CD45RA-Cy5 and CD57-PE and counterstained with LDS751. (PDF 7778 kb)
Example of Flow Cytometric Analysis (WMV 7276 kb)
Flow FISH in Fume Hood (WMV 3089 kb)
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Baerlocher, G., Vulto, I., de Jong, G. et al. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 1, 2365–2376 (2006). https://doi.org/10.1038/nprot.2006.263
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