Multiplexed approaches correlating mitochondrial health to cell health using microcapillary cytometry

Abstract Mitochondria are critical cellular organelles that play a fundamental role in cellular metabolism and oxidative stress and are well known to trigger multiple cell death pathways. The study of sequence of mitochondrial events as it relates to apoptotic/cell death events can provide critical insights into mechanism of cellular homeostasis, stress and death. Availability of rapid and simplified cytometric testing methods for evaluating mitochondrial changes, apoptosis and cell death in parallel can greatly enhance our understanding of mechanism of compound action. In this study, we investigated a series of compounds to evaluate apoptotic/cell death effects in context of mitochondrial changes using plate-based assays on Guava® easyCyte systems. Studies utilized multiplexed assays for mitochondrial membrane potential changes and apoptosis/cell death markers and allowed for easy identification of hit compounds. Dose and time response studies with Niclosamide, an anti-helmintic drug and comparison of effects with Gambogic acid and celastrol demonstrated early and significant mitochondrial impacts for niclosamide and gambogic acid. No apoptotic or cell death impacts were observed in parallel at low doses/short times of incubation for niclosamide, while increased time with niclosamide caused increase in mitochondrial, apoptotic and cell death response. The method demonstrates great power in being able to distinguish between potency of compounds and conditions in modulating mitochondrial/apoptotic changes. The simplicity of the assays described coupled with the ease of use of plate based microcapillary cytometry can provide researchers valuable tools to obtain a more comprehensive insight into how compounds modulate mitochondria and its relationship with subsequent apoptosis/cell death pathways.