Kahn E, Frouin F, Bruzzoni-Giovanelli H, Calvo F, Di Paola R, Linarès-Cruz G
Institut National de la Santé et de la Recherche Médicale U494, Centre Hospitalier Universitaire Pitié Salpêtrière, Paris, France.
Anal Quant Cytol Histol. 1999 Feb;21(1):1-7.
To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS).
For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations.
Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3.
It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.
