Flow cytometric determination of cell proliferation in hypertensive blood vessels

Autor: Andrew Hirsch, Smita Thakker-Varia, George J. Poiani, Edward J. Yurkow, Carol A. Tozzi, Arvind K. Das, David J. Riley
Rok vydání: 1999
Předmět:
Zdroj: Cytometry. 37:81-84
ISSN: 1097-0320
0196-4763
DOI: 10.1002/(sici)1097-0320(19990901)37:1<81::aid-cyto10>3.0.co;2-j
Popis: BACKGROUND Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. METHODS Pulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. RESULTS An approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling. CONCLUSIONS Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.
Databáze: OpenAIRE
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