Measurement of Cytogenetic Damage in Rodent Blood with a Single‐Laser Flow Cytometer

Stephen Dertinger1, Dorthea Torous1, Nikki Hall1, Carol Tometsko1

1 Litron Laboratories, Rochester, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.21
DOI:  10.1002/0471142956.cy0721s23
Online Posting Date:  February, 2003
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The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity. Double‐strand chromosome breaks or dysfunction of the mitotic spindle apparatus can lead to micronuclei formation in dividing cells. Erythrocytes have become the target population of choice, as precursor cells are continuously dividing and micronuclei are readily observable after extrusion of nuclei. The traditional method has been to stain peripheral blood or bone marrow smears and microscopically determine the frequency of micronucleated erythrocytes. Because these events are rare, the process is tedious and time consuming. This unit describes a procedure for fixing and staining rodent peripheral blood for flow cytometric enumeration. The combination of reagents provides for differential labeling and enumeration of four subpopulations: mature erythrocytes, micronucleus‐containing mature erythrocytes, young erythrocytes (reticulocytes), and micronucleus‐containing young erythrocytes. Malaria‐infected rodent erythrocytes, which closely mimic micronucleus‐containing erythrocytes, serve as a biological standard to facilitate rational and consistent equipment calibration. Keywords: flow cytometry; genotoxicity; chromosome damage; micronuclei; reticulocytes; propidium iodide; CD71‐defined antigen The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Mouse or Rat MicroFlowPLUS kit (Litron Laboratories) containing:
  •  Solution A (fixative; i.e., methanol)
  •  Solution B (anticoagulant)
  •  Solution C (washing/diluting buffer)
  •  Malaria Biostandard sample
  • Experimental rodent(s) (i.e., mouse or rat)
  • RNase/anti‐CD71‐FITC solution (see recipe)
  • PI solution (see recipe)
  • 15‐ml screw‐cap polypropylene tubes
  • −70° to −85°C (preferred) chest freezer
  • 2.5‐ml microcentrifuge tubes
  • Centrifuge (refrigerated preferred) with swinging‐bucket rotor
  • Polypropylene or polystyrene flow cytometry tubes
  • Foil
  • Flow cytometer:
  •  Excitation: 488 nm
  •  Filters: 530 ± 30‐nm band‐pass (green) and 650‐nm long‐pass (red)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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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., and Tometsko, K.R. 1996. Simple and reliable enumeration of micronucleated reticulocytes with a single‐laser flow cytometer. Mutat. Res. 371:283‐292.
   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.
   Donovan, J. and Brown, P. 1995. Blood collection. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, and W. Strober, eds.) pp. 1.7.1‐1.7.8. John Wiley & Sons, New York.
   Hayashi, M., Kodama, Y., Awogi, T., Suzuki, T., Asita, A.O., and Sofuni, T. 1992. The micronucleus assay using peripheral blood reticulocytes from mitomycin C‐ and cyclophosphamide‐treated rats. Mutat. Res. 278:209‐213.
   Hayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.‐D., Blakey, D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., and Sutou, S. 2000. In vivo rodent erythrocyte micronucleus assay: Aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring. A report from the International Workshop on Genotoxicity Test Procedures (IWGTP). Environ. Molec. Mutagen. 35:234‐252.
   OECD, 1997. Mammalian erythrocyte micronucleus test. In OECD Guidelines for the Testing of Chemicals, Section 4, Guideline 474. Organisation for Economic Cooperation and Development (OEDC), Paris.
   Seligman, P., Allen, R., Kirchanski, S., and Natale, P. 1983. Automated analysis of reticulocytes using fluorescent staining with both acridine orange and an immunofluorescence technique. Am. J. Hematol. 14:57‐66.
   Serke, S. and Huh, D. 1992. Identification of CD71 (transferrin receptor) expressing erythrocytes by mutiparameter‐flow‐cytometry (MP‐FCM): Correlation to the quantitation of reticulocytes as determined by conventional microscopy and by MP‐FCM using an RNA staining dye. Br. J. Haematol. 81:432‐439.
   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.
   Torous, D.K., Hall, N.E., Dertinger, S.D., Diehl, M.S., Illi‐Love, A.H., Cederbrant, K., Sandelin, K., Bolcsfoldi, G., Ferguson, L.R., Pearson, A., Majeska, J.B., Tarca, J.P., Hewish, D.R., Doughty, L., Fenech, M., Weaver, J.L., Broud, D.D., Gatehouse, D.G., Hynes, G.M., Kwanyuen, P., McLean, J., McNamee, J.P., Parenteau, M., Van Hoof, V., Vanparys, P., Lenarczyk, M., Siennicka, J., Litwinska, B., Slowikowska, M.G., Harbach, P.R., Johnson, C.W., Zhao, S., Aaron, C.S., Lynch, A.M., Marshall, I.C., Rodgers, B., and Tometsko, C.R. 2001. Flow cytometric enumeration of micronucleated reticulocytes: High transferability among 14 laboratories. Environ. Molec. Mutagen. 38:59‐68.
   Wakata, A., Miyamae, Y., Sato, S., Suzuki, T., Morita, T., Asano, N., Awogi, T., Kondo, K., and Hayashi, M. 1998. Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS‐MMS. Environ. Molec. Mutagen. 32:84‐100.
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