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Evaluation of the effects of cryopreservation of isolated erythrocytes and leukocytes of largemouth bass by flow cytometry (1995)

Fisher, S. K. ; Lingenfelser, J. T. ; Jagoe, C. H. ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

Journal of fish biology 46 (1995), S. 0

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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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1095-8649.1995.tb05983.x

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Flow cytometry in toxicity analysis (1990)

Dallas, C. E. ; Evans, D. L.

[s.l.] : Nature Publishing Group

Nature 345 (1990), S. 557-558

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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

URL: http://dx.doi.org/10.1038/345557a0

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Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis (1994)

Fisher, S. K. ; Dallas, C. E. ; Jagoe, C. ; [et al.]

Springer

Cell biology and toxicology 10 (1994), S. 145-153

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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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00757557

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