Technical Notes: A Simple Procedure for Long-Term Storage and Preparation of Fish Cells for DNA Content Analysis Using Flow Cytometry

Gold, J. R.; Ragland, Chara J.; Birkner, Matthias C.F.; Garrett, Gary P.

doi:10.1577/1548-8640(1991)053<0108:tnaspf>2.3.co;2

Cited by 10 publications

(9 citation statements)

References 3 publications

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“…After one‐, two‐, and three‐weeks exposure, organisms were prepped for flow‐cytometric analysis. The tadpole flow‐cytometric protocol was modified from Gold et al [21]. Tadpoles were homogenized in 10 ml of a 1 × phosphate buffered solution ([PBS], Sigma, St. Louis, MO, USA) and then filtered through a 53‐μm mesh.…”

Section: Methodsmentioning

confidence: 99%

Developmental impact of atrazine on metamorphing Xenopus laevis as revealed by nuclear analysis and morphology

Freeman

Rayburn

2005

Enviro Toxic and Chemistry

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Atrazine is one of the major surface water contaminants in the midwestern United States. Speculations have arisen on the potential effects of atrazine contamination to anuran larvae developing in these surface waters. In this study, Xenopus laevis tadpoles were exposed to environmentally relevant concentrations of atrazine. Nuclear and morphological endpoints were used to assess the effects of atrazine on developing X. laevis. Atrazine significantly affected metamorphing X. laevis after three-weeks exposure compared to controls as revealed by flow cytometric nuclear DNA analysis. The flow cytometric analysis was reflective of developmental effects. The number of nuclei per organism also was analyzed. Nuclei number was found to be associated with X. laevis development. Nuclei counting showed significant effects of atrazine after five-weeks exposure. A third endpoint, Nieuwkoop and Faber (NF) morphological staging, also demonstrated that atrazine significantly affected development after four weeks. Atrazine was found to alter the timing of metamorphosis of X. laevis using both nuclear analysis and gross morphology. The NF staging was found to be a sensitive assay to measure effects of development, whereas flow cytometry provided an impartial quantitative measure.

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Section: Methodsmentioning

confidence: 99%

Developmental impact of atrazine on metamorphing Xenopus laevis as revealed by nuclear analysis and morphology

Freeman

Rayburn

2005

Enviro Toxic and Chemistry

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“…The DNA content analysis protocol is a modification of the Gold et al (1991) fish cell protocol for flow cytometry. Tadpoles were homogenized in 10·ml of a 1ϫ phosphatebuffered saline (PBS; Sigma, St Louis, MO, USA) and then filtered through a 53·µm mesh.…”

Section: Methodsmentioning

confidence: 99%

Metamorphosis inXenopus laevisis not associated with large-scale nuclear DNA content variation

Freeman

Rayburn

2004

Journal of Experimental Biology

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presence of nuclei of variable fluorescence intensity within the unfixed nuclei. Upon optimum fixation, which has been speculated to result in more homogeneous chromatin conformation and to reduce staining artifacts, the nuclei were observed to have less fluorescence intensity variation. The differential fluorescence observed in this study is consistent with the hypothesis that large-scale intraindividual DNA variation is not associated with amphibian metamorphosis.

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“…Hawke, Louisiana State University, personal communication). Fixed tissue has been used for ploidy estimations (Burns et al 1986;Johnstone and Lincoln 1986;Gold et al 1991;Brown et al 2000;Lamatsch et al 2000), and development of a fixation protocol optimized for flow cytometric ploidy determination may allow longterm storage of eyeballs or cells from them. Probable steps would include injection of various fixatives at percentages per volume of vitreous humor into eyeballs or removal of cells into fixative.…”

Section: Asian Carp Ploidymentioning

confidence: 99%

Use of Eyeballs for Establishing Ploidy of Asian Carp

Jenkins¹,

Thomas²

2007

N American J Fish Manag

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Grass carp Ctenopharyngodon idella, silver carp Hypophthalmichthys molitrix, and bighead carp H. nobilis are now established and relatively common in the Mississippi and Atchafalaya rivers. Commercial fishers of Louisiana's large rivers report recurrent catches of grass carp, and the frequency of bighead carp and silver carp catch is increasing. Twelve black carp Mylopharyngodon piceus were recently captured from the Mississippi and Atchafalaya River system, and 10 were analyzed for ploidy. By using the methods described herein, all 10 fish were determined to be diploid. Such correct identifications of ploidy of feral Asian carp species, as well as other species, would provide science-based information constructive for meeting reporting requirements, tracking fish movements, and forecasting expansion of species distribution. To investigate the postmortem period for sample collection and to lessen demands on field operations for obtaining samples, a laboratory study was performed to determine the length of time for which eyeballs from postmortem black carp could be used for ploidy determinations. Acquiring eyes rather than blood is simpler and quicker and requires no special supplies. An internal DNA reference standard with a documented genome size, including erythrocytes from diploid black carp or Nile tilapia Oreochromis niloticus, was analyzed simultaneously with cells from seven known triploid black carp to assess ploidy through 12 d after extraction. Ploidy determinations were reliable through 8 d postmortem. The field process entails excision of an eyeball, storage in a physiological buffer, and shipment within 8 d at refrigeration temperatures (48C) to the laboratory for analysis by flow cytometry.

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Technical Notes: A Simple Procedure for Long-Term Storage and Preparation of Fish Cells for DNA Content Analysis Using Flow Cytometry

Cited by 10 publications

References 3 publications

Developmental impact of atrazine on metamorphing Xenopus laevis as revealed by nuclear analysis and morphology

Developmental impact of atrazine on metamorphing Xenopus laevis as revealed by nuclear analysis and morphology

Metamorphosis inXenopus laevisis not associated with large-scale nuclear DNA content variation

Use of Eyeballs for Establishing Ploidy of Asian Carp

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