Mitochondrial Assays

Mitochondria, often referred to as the “powerhouses of the cell,” are essential organelles that drive cellular energy production, maintain metabolic balance, and regulate key processes such as apoptosis, calcium signalling, and reactive oxygen species (ROS) homeostasis. Their unique biology, from their double membrane structure to their independent genome, has long intrigued researchers. Understanding mitochondria not only helps us decode fundamental cellular biology but also provides insights into diseases such as cancer, neurodegenerative disorders, and metabolic syndromes. Dysfunctional mitochondria are implicated in diseases such as Parkinson’s, Alzheimer’s, diabetes, and even aging. Targeting mitochondrial pathways offers potential therapeutic avenues, making robust analytical methods indispensable.

Figure 1. Mitochondria 

At o2h, we specialise in cutting-edge mitochondrial biology assays, helping researchers unravel the intricacies of these organelles and their impact on cellular health. Whether you need standardised assays or bespoke solutions tailored to your unique research needs, o2h offers unmatched flexibility and expertise.

High-Content Imaging (HCI) for Mitochondrial Biogenesis and Degradation

High-content imaging (HCI) enables a detailed and quantitative view of mitochondrial dynamics.

• Mitochondrial Biogenesis or Degradation (Mitophagy): Using fluorescent markers, such as COXI and TOMM20 we monitor the degradation of damaged mitochondria, providing insights into autophagy-related pathways. This assay allows for multiplex analysis, correlating mitochondrial changes with other cellular features, offering unparalleled depth for drug discovery and mechanistic studies.

Figure 2. Mitochondrial degradation assay. (A) Representative HCI images from nuclei, COXI and TOMM20 staining following addition of chloramphenicol. Chloramphenicol blocks the mitochondrial ribosome presenting COXI production, whilst nuclear-encoded TOMM20 remains constant. Images acquired at 10X magnification, scale bar 250 μM. (B) Drug response curve for the ratio of COX1/TOMM20 against increasing chloramphenicol.

Jess-Based Western Blotting for Protein Quantification

Traditional western blotting can be labour-intensive and semi-quantitative. Enter Jess, an automated capillary electrophoresis platform that transforms protein analysis.

• With Jess, we can precisely quantify mitochondrial proteins, such as SDHA and MFN2, involved in mitochondrial fission, fusion, and apoptosis.
• The system offers high sensitivity and reproducibility, ideal for detecting subtle changes in mitochondrial signalling pathways or protein expression levels.

Figure 3. Detection of mitochondrial proteins. (A) Lane images from automated western blotting using JESS for mitochondrial proteins MFN2 and SDHA in the presence of decoupling agent CCCP which stimulates mitophagy. Beta actin used as a loading control. (B) Quantified relative protein intensities from (A).

MitoSOX Flow Cytometry for Reactive Oxygen Species (ROS) Analysis

• Mitochondria are both sources and targets of ROS, playing dual roles in cellular signalling and oxidative damage.
• Using MitoSOX Red, a mitochondrial superoxide-specific fluorescent dye, we measure superoxide production in live cells.

Flow cytometry quantifies ROS levels with single-cell precision, enabling researchers to assess the impact of oxidative stress or antioxidant interventions on mitochondrial function.

Figure 4. Quantifying mitochondrial reactive oxygen species. (A) Overlayed histograms showing levels of mitochondrial superoxide radicals in HeLa cells upon exposure to respective treatments. (B) Representation of MitoSOX Red mean fluorescence intensity (MFI) with respect to vehicle control. (C) Representation of absolute MitoSOX Red fluorescence intensity in terms of Geometric mean. (D) Extent of MitoSOX Red position cells represented as percentage of parent population. As shown in the figure, serum starvation greatly induces mitochondrial superoxide radicals. FCCP and Oligomycin A also causes induction of mitochondrial ROS.

Calcium Signalling Assays

Mitochondria are the second most prominent reservoirs of intracellular Calcium and are therefore integral to calcium homeostasis, acting as buffers to modulate intracellular calcium levels.

Using the FLIPR (Fluorescence Imaging Plate Reader) system and the Calcium-binding dye RHOD-2 AM, we measure mitochondrial calcium uptake in response to pharmacological agents or stressors. RHOD-2 AM dye is preferentially taken up by mitochondria.

This high-throughput, real-time assay offers insights into calcium-mediated signalling pathways, crucial for understanding mitochondrial regulation and its systemic effects.

Figure 5. HeLa cells were stained with RHOD-2 AM dye before being subjected to treatment with ATP and FCCP with or without Thapsigargin (THP), an inhibitor of ER Calcium channels (Ca²⁺ ATPase), depletes ER calcium stores and increases cytosolic calcium levels. The fluorescence measurements were performed on a FLIPR Penta system (Molecular Devices). (A) The flux of mitochondrial calcium in a time-dependent manner. (B) The fluorescence data is represented as calcium within mitochondria relative to control levels. ATP induces a brief but sharp rise in mitochondrial calcium levels followed by a gradual decrease. On the other hand, FCCP, an ionophore which depolarizes mitochondrial membrane potential, completely depletes mitochondrial calcium reserves.

Driving Discovery with Precision Tools

By leveraging advanced techniques like HCI, Jess, Flow Cytometry, and FLIPR, we provide researchers with a comprehensive suite of tools to study mitochondrial biology. These assays are not only critical for understanding cellular physiology but also for identifying novel drug targets and evaluating therapeutic efficacy.

At o2h, we are committed to supporting mitochondrial research with innovative solutions and expert analysis. We excel in developing bespoke assays, designed from the ground up to address your unique scientific questions. With a team of experienced scientists and state-of-the-art technology, o2h can craft innovative solutions to accelerate your mitochondrial research. Together, we can unlock the secrets of these dynamic organelles and their far-reaching impact on human health.

To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.