Measurement of cytogenetic damage in rodent blood with a single-laser flow cytometer

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.