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Cross‐Species Genetic Toxicity Assessment Accomplished by Flow Cytometric Analysis of Blood
Publication Name:
Current Protocols in Toxicology
Unit Number:
Unit 2.14
DOI:
10.1002/0471140856.tx0214s36
Online Posting Date:
May, 2008 Abstract
The formation of micronuclei in blood cells has been an established indicator of genotoxicity for decades. Standard microscopy methods are time-consuming and lack the objectivity that fully automated methods can provide. The ability of flow cytometric technology to rapidly and objectively survey thousands of cells for micronuclei can significantly improve the value of this endpoint. In addition, since many more cells can be scored, and specific populations can be targeted, species that historically have been difficult to obtain micronucleus data from, such as humans, can now be readily evaluated using this methodology. This unit describes a procedure for fixation, staining, and analysis of blood samples using materials supplied in MicroFlow kits (Litron Laboratories) and a single-laser flow cytometer. This methodology provides a reliable, robust assessment of chromosome damage that is used in basic science research as well as drug-safety screening programs at large pharmaceutical and chemical companies. Curr. Protoc. Toxicol. 36:2.14.1-2.14.15. © 2008 by John Wiley & Sons, Inc.
Keywords: genotoxicity; chromosome damage; micronuclei; reticulocytes; erythrocytes; CD71-defined antigen; CD61-defined antigen; cross species
Materials
Basic Protocol: Genetic Toxicity Assessment in Mouse Blood
Materials
- MicroFlow
PLUS kit (Litron Laboratories) specific for the species in use and containing:- Fixative (solution A)
- Anticoagulant (solution B)
- Washing/diluent buffer (solution C), ice cold
- RNase solution (solution D)
- Anti-CD71-FITC (solution E)
- Propidium iodide (solution F)
- Anti-CD61-PE (solution G)
- Malaria biostandard
- Peripheral blood sample (e.g., mouse, rat, canine, nonhuman primate, human) treated according to experimental design
- Labeling solution I (see
- Labeling solution II (see
- DNA staining solution (see
- 2.5-ml microcentrifuge tubes
- 15-ml screw-cap polypropylene tubes
- –85°C chest freezer
- Refrigerated centrifuge
- 12 × 75–mm polypropylene or polystyrene flow cytometry tubes
- Aluminum foil
- Flow cytometer equipped with 488-nm excitation and appropriate filters (e.g., 530- ±30-nm band-pass filter and 650-nm long-pass filter for green and red fluorescence, respectively)
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Figures
-
Figure 2.14.1Set the Single cells region in Plot A of the template (A) and using Plot E, place the large population of the CD71-negative mouse blood sample just below the boundary of the RETs region (B) and low in the FL3 fluorescence channel. -
Figure 2.14.2A kit-supplied Malaria biostandard sample stained in solution I is used to maximize the fluorescent resolution of RETs from NCEs, as well as erythrocytes with and without a micronucleus-like DNA content (A). The Malaria biostandard is also used to set appropriate compensation. Appropriate FL3-%FL2 compensation is evident when parasitized cells exhibit a vertical profile (B). This will be observed in Plot E of the template. -
Figure 2.14.3The Malaria biostandard stained in solution I is used to set appropriate compensation of FITC (FL1) out of the yellow (FL2) channel (A). Appropriate FL3-%FL2 compensation is evident when RETs are directly above NCEs (B). This will be observed in Plot E of the template. -
Figure 2.14.4The Malaria biostandard stained in solution I is used to set appropriate compensation of propidium iodide (FL3) out of the yellow (FL2) channel (A). Appropriate FL2-%FL3 compensation is evident when parasitized cells are no longer evident in the platelets region (B). This will be observed in Plot D of the template. -
Figure 2.14.5Following appropriate adjustment of PMT voltages and compensation, the Malaria biostandard stained in solution II should exhibit a platelet staining pattern similar to this plot. This will be observed in Plot D of the template. -
Figure 2.14.6For the -
Figure 2.14.7Representative blood samples from control (A) and genotoxicant-treated (B) mice. Lower left quadrant = NCE; lower right quadrant = micronucleated NCE; upper left quadrant = RET; upper right quadrant = micronucleated RET. This will be observed in Plot F of the template.
Literature Cited
| Literature Cited | |
| Abramsson-Zetterberg, L., Grawe, J., and Zetterberg, G. 1999. The micronucleus test in rat erythrocytes from bone marrow, spleen and peripheral blood: The response to low doses of ionizing radiation, cyclophosphamide and vincristine determined by flow cytometry. Mutat. Res. 423: 113-124. | |
| Dertinger, S.D., Torous, D.K., Hall, N., Tometsko, C.R., and Gasiewicz, T.A. 2000. Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry. Mutat. Res. 464: 195-200. | |
| Dertinger, S.D., Torous, D.K., Hall, N., and Tometsko, C.R. 2003. Measurement of cytogenetic damage in rodent blood with a single-laser flow cytometer. Curr. Protoc. Cytometry 23: 7.21.1-7.21.9. | |
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| Honda, K.I., Ishiko, O., Hato, F., Kitagawa, S., Jikihara, I., Yoshida, H., and Ogita, S. 2001. CD71 antibody enhances iron uptake by mouse bone marrow cells and the survival potential of erythroid progenitor cells. Int. J. Mol. Med. 8: 135-140. | |
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| Tometsko, A.M., Torous, D.K., and Dertinger, S.D. 1993. Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model. Mutat. Res. 292: 129-135. | |
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| Tweats, D.J., Blakey, D., Heflich, R.H., Jacobs, A., Jacobsen, S.D., Morita, T., Nohmi, T., O'Donovan, M.R., Sasaki, Y.F., Sofuni, T., and Tice, R.; IWGT Working Group. 2007. Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards. Mutat. Res. 627: 78-91. | |
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