A Simple, Robust Automated Multiplexed Cell-Based Assay Process for the Assessment of Mitochondrial Dysfunction and Cytotoxicity
Poster Feb 16, 2012
Brad Larson, Peter Banks, Tracy Worzella, Andrew Niles and Timothy Moeller
This poster demonstrates the utility of a multiplexed assay to assess cell membrane integrity changes (cytotoxicity), as well as mitochondrial function (ATP levels). Cytotoxicity is first assessed by measuring a distinct protease activity associated with necrosis using a fluorogenic peptide substrate (bis-AAF-R110) to measure “dead cell protease activity”. The substrate cannot cross the intact membrane of live cells and therefore gives no signal with viable cells. Mitochondrial function is then measured by adding an ATP detection reagent, resulting in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The two assay readouts used together can distinguish between compounds that exhibit mitochondrial toxicity versus overt cytotoxicity. Mitochondrial toxicity will result in a decrease in ATP production with little to no change in membrane integrity. A cytotoxic effect, such as primary necrosis, will also show a decrease in ATP but in conjunction with loss of membrane integrity. In addition, the multiplexed nature of the assay decreases data variability that could be seen when running these two assays sequentially.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE
15th International Conference on Surgical Pathology and Cancer Diagnosis
Apr 15 - Apr 16, 2019