The JAM-assay: optimized conditions to determine death-receptor-mediated apoptosis

Apoptosis induced by interaction of members of the TNF-/TNF-receptor superfamily has been considered as a major mechanism of cell-mediated cytotoxicity. For functional analysis, the 51Cr release assay has been widely used, which requires loss of membrane integrity in the apoptotic target cell. However, loss of membrane integrity is a late event during apoptosis and therefore only late apoptotic cells will be detected by this method. In contrast, the JAM-assay first described by Polly Matzinger has been demonstrated to be more sensitive than the 51Cr release assay, since this method is dependent on DNA-fragmentation which precedes loss of membrane integrity in most apoptotic cells. The JAM-assay is easier to perform, less expansive, and safer than the current standard (51)Cr release assay. Therefore, this article will focus on optimized conditions of the JAM-assay to detect and quantitate Fas (CD95/Apo-1)-induced apoptosis as an example of death-receptor-mediated cytotoxicity.