The cytochrome c release assay is a laboratory procedure that detects a definitive event in programmed cell death, also known as apoptosis. The assay determines if a cell has initiated self-destruction by measuring the location of a protein called cytochrome c.
When this protein moves from its normal location into the main cellular fluid, it signals that the cell is irreversibly committed to dying. This provides a clear snapshot of a cell’s fate, indicating whether it has received an internal instruction to self-destruct.
The Role of Cytochrome C in Cell Death
Under normal conditions, cytochrome c resides within the mitochondria, where it is part of the electron transport chain. It acts as an electron shuttle, a process necessary for creating adenosine triphosphate (ATP), the cell’s main energy source. Cytochrome c is bound to the inner mitochondrial membrane, facilitating electron movement between two protein complexes, known as Complex III and Complex IV.
The cell also possesses a highly regulated mechanism for self-elimination called apoptosis. This controlled process is activated in response to specific triggers, such as irreparable DNA damage. The release of cytochrome c from the mitochondrial intermembrane space into the main cellular fluid, the cytosol, is a decisive step in this pathway.
Once in the cytosol, cytochrome c binds to a protein called Apoptotic protease-activating factor-1 (Apaf-1). This interaction leads to the formation of a large protein complex known as the apoptosome. The apoptosome then activates a family of enzymes called caspases, which act as the executioners of apoptosis by systematically breaking down cellular components.
Fundamental Steps of the Assay
The first step is to separate the cell’s components to isolate the mitochondria and the cytosol. This begins with cell lysis, where the outer membrane is gently ruptured while leaving internal organelles like the mitochondria intact. This can be done using buffers with mild detergents or by mechanical means, such as a Dounce homogenizer.
The resulting mixture, or lysate, then undergoes differential centrifugation to separate contents by size and density. A low-speed spin pellets heavier components like the cell nuclei and any unbroken cells. The remaining liquid, or supernatant, is collected for the next step.
This supernatant, containing mitochondria and cytosolic proteins, is spun again at a high speed. The force pellets the dense mitochondria, leaving the cytosolic fraction as the remaining liquid. Researchers now have two separate samples: the mitochondrial pellet and the cytosolic supernatant, which contains any released cytochrome c.
Common Methods for Detection
Western blotting is a common detection method. It separates proteins from both the cytosolic and mitochondrial samples by size on a gel. The proteins are then transferred to a membrane and probed with an antibody that specifically binds to cytochrome c. In a healthy cell, a band for cytochrome c appears only in the mitochondrial sample, while in an apoptotic cell, the band appears in the cytosolic sample.
Another method is the Enzyme-Linked Immunosorbent Assay (ELISA), a quantitative technique performed in a multi-well plate. Wells are coated with a “capture” antibody that traps cytochrome c from the sample. A second, enzyme-linked antibody is added, which produces a color change whose intensity is directly proportional to the amount of cytochrome c present.
Immunofluorescence microscopy provides visual confirmation without cell fractionation. Cells are treated with a fluorescently labeled antibody that targets cytochrome c. Under a microscope, healthy cells show a distinct, punctate pattern where the fluorescence is confined to the mitochondria. Apoptotic cells display a diffuse pattern, with the signal spread throughout the cytosol, visually demonstrating the protein’s release.
Interpreting Assay Results
The interpretation is straightforward: the protein’s location determines the cell’s fate. A positive result is the detection of cytochrome c in the cytosol. This is a definitive indicator that the cell has committed to apoptosis, often called the “point of no return,” as it confirms the intrinsic apoptotic pathway has been activated.
To ensure results are valid, experiments include control samples. Untreated cells act as a negative control, where cytochrome c should be found only in the mitochondrial fraction. This comparison allows researchers to conclude that the substance or condition being tested is directly responsible for initiating apoptosis.
The assay has significant applications in cancer research and drug development. A primary goal is to find drugs that selectively induce apoptosis in cancer cells. Scientists use this assay to test if a potential drug causes cytochrome c release in cancer cells, providing strong evidence of its effectiveness at triggering the cell death program.