Protocol

Apoptosis Methods and Protocols

Volume 282 of the series Methods in Molecular Biology pp 85-101

Immunoassay for Single-Stranded DNA in Apoptotic Cells

  • Oskar S. FrankfurtAffiliated withUniversity of Miami Medical School and Apostain Inc.

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Abstract

The apoptosis assay described in this chapter is based on the selective denaturation of DNA in condensed chromatin of apoptotic cells and the detection of denatured DNA with a monoclonal antibody highly specific to single-stranded DNA. Optimal results are obtained by the heating at a relatively low temperature in the presence of formamide. The assay detects apoptotic cells but not necrotic cells or cells with DNA breaks in the absence of apoptosis. The sensitivity of the assay reflects the detection of early and late apoptosis. Apoptotic cells are detected in the sections of frozen or formalin-fixed paraffin-embedded tissues by immunohistochemistry and in the cell suspensions by flow cytometry or fluorescence microscopy. Apoptosis enzyme-linked immunoassay based on DNA denaturation by formamide in microtiter plates and one-step immunostaining is applied for high-throughput screening of drugs. The enzyme-linked immunoassay has the ability to distinguish anticancer drugs from toxic chemicals, to predict selective toxicity to cancer cells, and to detect drug synergism.

Key Words

Apoptosis single-stranded DNA ELISA drug screening monoclonal antibody immunohistochemistry flow cytometry