Last update:
12-Jul-2004

The localization of apoptotic cells, which was previously based on morphology and the detection of DNA fragmentation by biochemical or histochemical techniques, is now performed by more specific enzymatic methods for the in situ visualization of fragmented DNA. These methods rely on the use of DNA polymerase I (ISNT, in situ Nick Translation) or TdT (TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling). The basic principle is the insertion or addition of labeled (biotinylated) nucleotides in sites of single- and/or double-strand breaks. The DNA fragmentation techniques are applied to frozen or paraffin-embedded tissue sections. Long fixation times should be avoided because they reduce sensitivity. Factors such as autolysis, fixation, embedding, sectioning, quenching of endogenous peroxidase and necrosis may cause "nonapoptotic" DNA fragmentation. Pretreatment procedures include microwaving and/or predigestion with pepsin or proteinase K. For validating the technique the use of appropriate positive and negative control tissue samples is indispensable, and the multiparametric approach, i.e. TUNEL and morphological or immunohistochemical data, is applied for detection of apoptosis-related proteins, or in situ hybridization. Antisera against activated caspase-3 are considered to be specific markers for apoptotic cells because no activation of the caspase cascade has been found in necrotic cell death. Annexin V is used for flow-cytometric detection of apoptotic cells; it binds to phosphatidylserine transposed to the outer membrane leaflet during the apoptotic process. Phosphatidylserine externalization precedes DNA fragmentation, thus allowing the detection of apoptotic cells in the early stages of apoptosis. Comet assay detects fragmented DNA at the single cell level by electrophoresis. The technique was named according to the characteristic shape seen when the DNA exits the nucleus and cell body and migrates towards the anode. Different conditions allow the study of either single- or double-strand DNA breaks. The foremost advantage of the comet assay is that it measures the response of individual cells, thus allowing one to study heterogeneity within a cell population. Combination of the results of the above mentioned techniques permits the detailed and extensive analysis of cell death phenomenon and allows the quantification of the amount of fragmented DNA and cellular response to DNA damage.

Key words: Apoptosis, Caspases, Comet, Fragmented DNA, Tunel.