Telomere length measurements in leukocyte subsets by automated multicolor flow-FISH

Background: 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 rep eats 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. (C) 2003 Wiley-Liss, Inc.