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Methods in Cell Physiology

Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon

¹Graduate Group in Molecular and Biochemical Nutrition, University of California, Berkeley 94720; and ²Division of Endocrinology and Metabolism, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94141

Submitted 9 April 2003 ; accepted in final form 22 January 2004

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and ²H₂O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to ²H₂O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from ²H₂O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis.

carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes

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