Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptoti c cells by means of fluorescence using TdT-mediated dUTP-biotin nick end la beling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and fa ctor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a m odel. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanat e (TRITC). The DNA from cell nuclei teas counterstained using chromomycin A 3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the abilit y to detect DNA breaks in individual cells using TUNEL techniques and its a mplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of i mages of TUNEL preparations and on four-dimensional (4-D) sequences of imag es of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characte rized on dynamic sequences of images. Characterization was improved when FA MIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum gf TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNE L methods can be used to visualize and localize multiple DNA breaks in lymp hoid reverted cells in improved methods of experimentation.