Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells.
Anal Quant Cytol Histol
; 21(1): 1-7, 1999 Feb.
Article
in En
| MEDLINE
| ID: mdl-10068768
ABSTRACT
OBJECTIVE:
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). STUDYDESIGN:
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.RESULTS:
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.CONCLUSION:
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.
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Database:
MEDLINE
Main subject:
Microscopy, Confocal
/
In Situ Nick-End Labeling
/
DNA Fragmentation
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Year:
1999
Type:
Article