Table 1.
Summary of the in organello assays for mitochondrial quality assessment, viability and functions.
| S. No. | Assay Name | Assay Type | Information Provided |
|---|---|---|---|
| Quality Control Procedure | |||
| 1. | Electron microscopy | Transmission and Scanning electron microscope (TEM and SEM) | Mitochondrial enrichment/yield and morphological integrity |
| 2. | Mitochondrial stains | Spectroscopy-, Flow cytometry-, or Fluorescence microscopy-based | Mitochondrial enrichment and integrity |
| 3. | Marker proteins | Immunoblotting | Enrichment |
| 4. | Lactate dehydrogenase activity assay | Spectroscopy-based | Mitochondrial enrichment/yield and integrity |
| 5. | Cytochrome c release | Immunoblotting or spectroscopy-based | Mitochondrial enrichment/yield and integrity |
| 6. | Oxidation of exogenous ascorbate/ cytochrome c |
Spectroscopy-based or polarographic assay | Mitochondrial intactness |
| 7. | Succinate dehydrogenase (Complex II) activity assay | Spectroscopy-based | Mitochondrial enrichment/yield and integrity |
| 8. | Citrate synthase activity assay | Spectroscopy-based | Mitochondrial enrichment/yield and integrity, and as a normalization control |
| 9. | Fumarase activity assay | Spectroscopy-based | Mitochondrial integrity |
| 10. | Adenylate kinase activity assay | Spectroscopy-based | Mitochondrial integrity |
| 11. | Glutamate dehydrogenase activity assay | Spectroscopy-based | Mitochondrial integrity |
| 12. | Calcium buffering capacity | Fluorimetric | Mitochondrial integrity |
| Functional and Bioenergetic Assays | |||
| 1. | Mitochondrial membrane potential (cationic fluorescent dyes) | Fluorimetric, fluorescence spectroscopy- or cell cytometry-based | Mitochondrial integrity, viability and bioenergetic capacity |
| 2. | Ca2+-induced swelling (mitochondrial permeability transition) | Light scattering (spectroscopy)-based | Mitochondrial integrity, viability and bioenergetic capacity |
| 3. | Calcium buffering capacity (Ca sensitive fluorescent dyes) |
Fluorescence spectroscopy-based | Mitochondrial integrity and viability |
| 4. | Electron transport chain complexes activity assays - Complex I - Complex II - Complex I-III - Complex II-III - Complex IV - Complex V |
Spectroscopy-based, polarographic, or bioluminescence-based assay |
Mitochondrial viability and bioenergetic capacity |
| 5. | Oxygen consumption | Polarographic or fluorescence spectroscopy-based | Mitochondrial viability and bioenergetic capacity |
| 6. | Tricarboxylic acid cycle enzyme activity assays | Spectroscopy-based | Mitochondrial viability and bioenergetic capacity |
| Assays for Oxidative Damage and Redox Homeostasis | |||
| 1. | Dichlorofluorescein (DCF) assay | Fluorescence spectroscopy-based | Amounts of oxidative species |
| 2. | Superoxide/peroxide production | Spectroscopy- or fluorescence spectroscopy-based | Production of superoxide anion and hydrogen peroxide |
| 3. | Griess assay | Spectroscopy-based | Amounts of nitric oxide end products |
| 4. | Lipid peroxidation | Spectroscopy-based | Lipid oxidation |
| 5. | Protein carbonylation | Immunoblotting or spectroscopy-based | Protein oxidative damage |
| 6. | Protein thiol oxidation | Spectroscopy-based | Protein oxidative damage |
| 7. | Antioxidant capacity | Spectroscopy-based | Free radical scavenging |
| 8. | Glutathione (GSH) signalling - Reduced GSH levels - Glutathione reductase - Glutathione peroxidase - Glutathione S-transferase |
Spectroscopy- or fluorescence spectroscopy-based |
Antioxidant GSH signalling |
| 9. | Activity assay for superoxide dismutase | Spectroscopy- or fluorescence spectroscopy-based | Superoxide scavenging |
| Protein Translation Assays | |||
| 1. | Radiolabeled cysteine/methionine incorporation assay | Liquid scintillation or gel autoradiograhy | De novo protein translation |
| 2. | Puromycin incorporation assay | Immunoblotting | De novo protein translation |