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Am J Physiol Cell Physiol (February 11, 2004). doi:10.1152/ajpcell.00139.2003
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Submitted on April 9, 2003
Accepted on January 22, 2004

Isolation of Nuclei from Label-Retaining Cells and Measurement of Their Turnover Rates in Rat Colon

Sylvia J Kim1, Sandy Cheung1, and Marc K Hellerstein2*

1 Graduate Group in Molecular and Biochemical Nutrition, University of California at Berkeley, Berkeley, CA, USA
2 Graduate Group in Molecular and Biochemical Nutrition, University of California at Berkeley, Berkeley, CA, USA; Department of Medicine, University of California at San Francisco, San Francisco, CA, USA

* To whom correspondence should be addressed. E-mail: march{at}nature.berkeley.edu.

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 bromodeoxyuridine (BrdU) and 2H2O. Male Fisher 344 rats were given BrdU in drinking water continuously for 2-8 weeks. The BrdU was then discontinued (BrdU wash-out) and animals (n=33) were switched to 2H2O in drinking water and sacrificed after 2,4 and 8 weeks. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percent of LRCs were 7% and 3.8% after 4 and 8 weeks of BrdU wash-out, respectively. Turnover rates of LRCs were measured based on 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.




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