DYNAMIC FUNCTIONAL AND STRUCTURAL-ANALYSIS OF LIVING CELLS - NEW TOOLS FOR VITAL STAINING OF NUCLEAR-DNA AND FOR CHARACTERIZATION OF CELL MOTION

Increasing interest has been paid to applications of fluorescence meas urements to analyze physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization as well as cell moti on during the cell cycle. This approach involves both optimal conditio ns for DNA staining and cell tracking methods. In this context, this r eport describes a stoichiometric method for nuclear DNA specific stain ing, using the bisbenzimidazole dye Hoechst 33342 associated with vera pamil, a calcium membrane channel blocker. This method makes it possib le to correlate variations of nuclear DNA content with cell motion in cells that are maintained alive. Motion measurement is the second goal of this paper and it explains the snake-spline method, and the associ ated cell following method.