CELL CYCLE ANALYSIS VIA BRDU HOECHST FLOW CYTOMETRY PRINCIPLES AND APPLICATIONS

Research on oncogenes and growth factors has immensely improved our understanding of the regulation of cellular proliferation, and both areas of research have recently converged. 1 , 2 In contrast to these major conceptual advances, the methodology of measuring the cellular response to mitogens has lagged behind, relying mostly on radioactive precursor uptake and dilution studies introduced into biomedical research over 40 years ago. These conventional methods of measuring the cellular mitogen response are complicated by problems due to catabolism and undetermined pool sizes of the radioactive precursor; 3 in the case of autoradiographic analysis, the time and labor involved in the analysis is often extensive. Two examples may suffice to illustrate these drawbacks; in mammalian cells, 3H-thymidine incorporation affects cell cycle progression and leads to arrest in the G2 phase of the cell cycle which is donor-age dependent. 4 In the slime mold, Physarum polycephalum, 50% of the macroplasmodial nuclei display label by autoradiography, but only 10% incorporation is found in acid-precipitable material at identical times after mitosis. 5 Some of the drawbacks of the radioisotope methods have been overcome by the introduction of flow-fluorometric assessment of DNA content as a measure of cell activation and cell cycle progression. 6–8 By combining DNA with RNA measurements, flow cytometry achieved an unprecedented resolution of the kinetic events associated with cell activation. 9 , 10 The recent merging of flow cytometry with immunocytochemistry has added yet another technical dimension: bromo- or iododeoxyuridine incorporated during semiconservative replication is detected by monoclonal antibodies, permitting a clear distinction between replicating and nonreplicating cells, as well as determinations of cell cycle traverse rates. 11–15 Current technical improvements are likely to increase the utility of the halogenated nucleoside antibody assays. 16 , 17 Moreover, antibodies against activation- and replication-specific nuclear antigens (such as Ki 67 or Cyclin/PCNA) may prove useful in the near future. 18 , 19.