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Method of Specific Detection of Apoptosis Using Formamide-induced DNA Denaturation Assay

Yuko Ito, Masa-Aki Shibata, Ken Kusakabe and Yoshinori Otsuki

Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Osaka, Japan

Correspondence to: Prof. Yoshinori Otsuki, Department of Anatomy and Cell Biology, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan. E-mail: an1001{at}art.osaka-med.ac.jp

We compared the reliability between apoptosis detection methods, namely, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method and formamide-induced DNA denaturation assay using a monoclonal antibody (MAb) to single-stranded DNA (ssDNA) (formamide–MAb assay). Reaction targets in these methods are different: the TUNEL method recognizes free 3'-OH DNA ends, whereas the formamide–MAb assay detects ssDNA itself (25–30 bp). We found that the formamide–MAb assay immunohistochemically detected apoptotic cells, whereas the TUNEL method detected apoptotic cells as well as mitotic and necrotic cells. The TUNEL method recognized not only 3'-OH DNA ends cleaved by DNase during apoptosis but also constitutive physiological nicking that occurs in DNA duplication and histone posttranslational modifications during mitosis and random DNA breaks during necrotic execution. By electron microscopy, the mean labeling density (the number of 3'-OH DNA ends/nuclear area) obtained by the TUNEL method was determined to be consistently higher than that (the number of ssDNAs/nuclear area) obtained by the formamide–MAb assay. On the basis of these findings, we conclude that the formamide–MAb assay was more specific than the TUNEL method for the detection of apoptotic cells using electron microscopy; however, the labeling intensity of the formamide–MAb assay was slightly weaker than that of the TUNEL method. (J Histochem Cytochem 54:683–692, 2006)

Key Words: single-stranded DNA • formamide • denaturation • TUNEL method • apoptosis • necrosis • mitosis

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