Use of Diphenylamine in the Detection of Apoptosis

Abstract

The diphenylamine assay is a very useful tool in measuring apoptosis by determining the percentage of fragmentation of known amounts of DNA into oligosomal-sized fragments. Another advantage of the diphenylamine assay is that apoptotic DNA fragmentation can be analyzed in both adherent and shed cells following treatment or experimentation. Data obtained from the experiment are expressed as a percentage relative to uninduced or untreated controls. This assay was first described by Dische (1,2) in the 1930s and later modified by Burton (3) in the mid-1950s. These modifications have resulted in an enhanced sensitivity of up to five times by adding sulfuric acid and acetaldehyde, and by allowing the colorimetric reaction to develop overnight at room temperature. These changes have also resulted in a reduction of the interference from other substances that were an initial drawback with the originally described method, further enhancing the sensitivity of the assay. The diphenylamine reaction takes advantage of the bonds between purines and deoxyribose, which are very labile. Once these bonds are broken, inorganic phosphates are liberated from the DNA and provide the substrate, which is measured by the reaction. The overall preparation time is approximately 3 h for 30 samples with incubation occurring overnight for 12 to 16 h. Reading of the results takes approximately 1 min per sample. This assay has been used to detect apoptotic fragmentation by others, and more recently by our group to evaluate cisplatin and Taxol induced fragmentation in ovarian carcinoma cell lines (4).