ApoTox-Glo Triplex Assay from Promega

The ApoTox-Glo Triplex Assay is a recent addition to a number of elegant kits released by Promega over the last year. It addresses the accurate quantification of apoptosis and cell death. The Triplex Assay name is derived from combining the strengths of three chemistries; the first measures cell viability, the second quantifies cytotoxicity and the third assays caspase activation, all for the same sample in a single well. Cell viability and cytotoxicity are calculated simultaneously by measuring the activity profiles of two protease markers in the first part of the assay. In the second part, caspase activity is determined by providing a luminogenic caspase substrate, allowing the quantification of apoptosis. The kit is provided with an internal control which enables the normalization of data by calculating the ratio of live cells to dead cells, independent of cell number. This is a significant advantage offered by the Triplex Assay because traditional in-house methods of quantifying cell death in culture requires adjustment of the starting material to accurately quantify between samples. This often involves collecting cells and then spinning and re-suspending specific amounts to extract meaningful results. Using the Triplex Assay, direct cell aliquots can be taken which greatly contributes to the efficiency and appeal of Promega’s assay.

Literature provided with the kit as well as conversations with our Promega sales representative informed us that the Triplex Assay had not been tried on plant cell samples. We were very keen to be amongst the first laboratories who would attempt the optimization of this assay on cell cultures of the model plant organism Arabidopsis. Working with the 96-well format of the Triplex Assay was very conducive to establishing a robust experimental design which would enable us to incorporate crucial negative and positive controls and study the effects of various treatments on our cell cultures. The substrates, reagents and buffers provided with the assay are very sensitive to light and temperature fluctuations, as well as dust and other particulate contamination, since these can interfere with the fluorescence reading. Therefore, it is important to work in a laminar flow hood, preferably in a controlled temperature environment and prevent extended exposure to light. After addition of the different protease and caspase substrates, the assay requires a 30 minute incubation at 37°C. I would recommend incubating for at least an hour to increase the sensitivity of the assay. We also found it important to analyze our data after plotting it as log-transformed data to account for the scale differences in cytotoxicity and cell viability measurements. Our results with plant cell cultures were very informative, highly sensitive and immensely encouraging!