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.
Hoves
Sabine
S
Department of Internal Medicine I, The University of Regensburg, 93042 Regensburg, Germany.
Krause
Stefan W
SW
Schölmerich
Jürgen
J
Fleck
Martin
M
D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D053938:DNA Fragmentation; D003713:Dendritic Cells / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism
D018124:Receptors, Tumor Necrosis Factor; D019014:fas Receptor; D013936:Thymidine