The Mito Stress Test (ACU Format) is designed to measure oxygen consumption rate (OCR) and extracellular acidification (ECA) simultaneously and in real time under tightly controlled atmospheric conditions using open-flow respirometry.

The assay was designed to work exclusively with microplate readers equipped with an Atmospheric Control Unit (ACU). BMG LABTECH’s multifunctional CLARIOstar® microplate reader with dual injection ports, time-resolved fluorescence detection, and an ACU was used to optimize the assay protocol. The ACU integrated into the CLARIOstar® reader facilitates measurement of cellular oxygenation at a controlled [O2], while phosphorescent extracellular O2 (MitoXpress® Xtra) and fluorescent extracellular pH (pH-Xtra™) probes provided in the kit enable multi-parametric detection of electron transport chain activity and glycolytic flux.

Enhanced assay performance with the CLARIOstar® ACU

By combining these advanced instrumentation and assay technologies, this kit eliminates the often confounding hyperoxic conditions under which most commercially available, cell-based in vitro assays are conducted. “Because ‘normoxia’ varies between tissue and cell type, Cayman’s kit provides a more physiologically relevant model for certain cell lines or primary cells that typically would not be exposed to ambient oxygen concentrations,” said Dr. David Hoffman, the lead developer of this kit. Rather than using a typical microchamber, the kit provides an alternative platform that relies on the difference in steady-state oxygen, which allows measurements of OCR and ECA under various oxygen concentrations that are controlled by the user.

“The fact that cells of internal tissues are not exposed to the ambient O2 tension has often been neglected for the sake of practical work and ease of use in the lab,” said Carl Peters, Application Specialist with BMG LABTECH, “This collaboration provides an easy-to-set-up system whereby physiological conditions can be effortlessly reproduced in the lab.” The ACU can fully regulate both [O2] and [CO2] at a range from 0.1–20%, which provides a means to measure mitochondrial function at atmospheric concentrations that are lower than ambient conditions. Pre-defined scripts to automatically control the CLARIOstar® are included in the kit protocol, making assay performance and subsequent data analysis an easy plug-and-play and walk-away experience. Assay results are determined based on the lifetime measurements of MitoXpress® Xtra and pH-Xtra™ in response to changes in O2 and pH. One-click assay calculation templates using BMG LABTECH’s MARS software interface make the process of data analysis nearly instant. View example data (PDF).

Cayman Chemical plans to develop and optimize additional assays for bioenergetics research utilizing the technology and software of the CLARIOstar®.