Heather Mary Brown, PhD | Application Scientist | Enzo Life Sciences
Matthew Hammer | Applications Scientist | Molecular Devices
Cheryl L. Bell, PhD | Field Applications Scientist | Molecular Devices


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MITO-ID® Membrane potential detection kit
HOECHST 33342

INTRODUCTION

From studying disease to evaluating drug and environmental compoundinduced toxicity, cell-based assays aimed at monitoring cellular health often focus on mitochondria. Mitochondrial function is a critical indicator of overall cell health, highlighted by the association of mitochondrial dysfunction and a variety of diseases including Parkinson’s Disease, Alzheimer’s Disease, heart failure, ischemic diseases, and cancer. Additionally, the pathogenesis of rare diseases can be driven by mutations in genes coding for mitochondrial proteins or in mitochondrial DNA.

Evaluating mitochondrial function is critical throughout drug development and testing. This is exemplified by the development of therapeutics to restore mitochondrial function, and the development of chemotherapeutics designed to target mitochondrial dysfunction in cancer. Furthermore, assessment of mitochondrial function is a vital step in preclinical drug safety assessment since mitochondrial dysfunction can be triggered by drugs and in turn, result in adverse effects on cellular and tissue health.

One sensitive and informative method to assess mitochondrial function is measuring mitochondrial membrane potential (MMP) to monitor changes in the metabolic activity of mitochondria. Mitochondrial membrane potential is a crucial tool for the detection of mitochondrial depolarization during apoptosis, the testing of multi-drug resistant cells, and the assessment screens. Evaluation of MMP is commonly performed with the use of lipophilic, cationic fluorescent dyes. The MITO-ID® membrane potential dye exhibits changes in its fluorescence depending on the status of mitochondrial membrane potential. Specifically, it exists as a greenfluorescent (FITC, 530 nM) monomer in the cytosol regardless of MMP, and aggregates inside healthy, energized mitochondria to emit orange-fluorescence (TRITC, 570 nm). Upon disruption of MMP, the MITO-ID dye exits the depolarized mitochondria, resulting in low tone orange fluorescent aggregates and an accumulation of the dye monomers in the cytosol.

We performed two mitochondrial toxicity assays utilizing the MITO-ID dye to assess changes in mitochondrial membrane potential in response to compound treatment. Assays were performed on a suspension cancer cell line, SJK, and an adherent cancer cell line, U2OS, to demonstrate the utility of MITO-ID dye to assess MMP in multiple cell types and the robust imaging and analysis capabilities of the ImageXpress Pico® Automated Cell Imaging System.

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