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  • 1
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 345 (1990), S. 557-558 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] The well-established clinical research tool of flow cytometry has now been applied to the detection of toxic injury to the cells of organisms exposed to environmental contaminants ...
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1095-8649
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We examined the effects of separation and freezing on fish leukocyte and erythrocyte morphology by light microscopy and on DNA content as measured by flow cytometry (FCM). Leukocytes and erythrocytes of largemouth bass Micropterus salmoides were isolated by density gradient centrifugation of whole blood, and frozen in liquid nitrogen in a buffer containing DMSO as a cryopreservative. The coefficient of variation (CV) of the G0/G1 peak of the cells was used to assess variation in nuclear DNA content within cell populations before and after separation and freezing treatments. In erythrocytes, the CV did not change significantly (P〉0.05) when nuclei were isolated and stained without freezing or when erythrocytes were frozen prior to nuclear isolation and staining. In leukocytes, freezing and thawing prior to isolation and staining of nuclei significantly increased the CV (P〈0.05), and produced hyperdiploid shoulders of the G0/G1 peak. However, the CV of leukocyte nuclei that were isolated and stained prior to freezing and the CV of non-frozen leukocyte nuclei did not differ (P〉0.05). Microscopy showed that the freezing protocol had little effect on erythrocyte morphology, but caused irregular swelling in leukocytes. Freezing intact leukocytes also significantly (p〈0.05) altered the apparent distribution of cells among the phases of the cell cycle as measured by FCM. The distributions of leukocyte nuclei that were isolated and stained prior to freezing were not different to non-frozen leukocytes. DNA measurements of nucleated blood cells are widely used in physiological, genetic and toxicological studies. Our results suggest that whole blood and erythrocytes for use in such studies can be frozen whole using a simple protocol, but leukocyte nuclei must be isolated and stained before freezing to avoid serious artifacts.
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  • 3
    ISSN: 1573-6822
    Keywords: DNA ; flow cytometry ; genotoxicology ; largemouth bass ; red blood cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Analysis of cellular DNA content by flow cytometry has been used to detect genetic changes associated with exposure to environmental contaminants. In lower vertebrates, nucleated red blood cells can be collected for analysis without harm to the animal. Because erythrocytes sampled from an individual should have identical amounts of DNA, the coefficient of variation (CV) around the G0/G1 peak should be small. Increases in CV can indicate genetic aberrations, but may also be caused by sample handling and preparation or problems with instrumentation. To increase confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument parameters should be identified and minimized. We assessed the effects of various sampling and handling protocols on the CV of nucleated blood cells collected from largemouth bass (Micropterus salmoides). We also compared the distribution of cells among the G0/G1, S, and G2/M phases of the cell cycle to see whether these were affected by sampling or treatment protocols. Groups of 7 fish were bled on 7 consecutive days, and blood from each fish was analyzed by flow cytometry when freshly collected, and after freezing for 1 hour or 10 days. The same fish were bled again over a consecutive 7-day period, and the experiment was repeated. CV and cell cycle distribution were not affected by our freezing protocol. Repeat sampling from the same individual did not affect CV, but altered the distribution of cells in the cell cycle, suggesting increased hemopoiesis in response to blood sampling. Day-to-day variation in the CV occurred in both fresh and frozen samples, probably as the result of small variations in instrument adjustments. These results demonstrate the suitability of this freezing protocol for these blood samples, and illustrate the importance of assessing sources of variation when using flow cytometry to screen wild populations in genotoxicological studies.
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  • 4
    Publication Date: 1990-06-01
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Published by Springer Nature
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  • 5
    Publication Date: 1994-06-01
    Print ISSN: 0742-2091
    Electronic ISSN: 1573-6822
    Topics: Biology , Medicine
    Published by Springer
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