Overview
Apoptosis or programmed cell death occurs when a cell has received sufficient damage to curtail steady functioning, but before ATP production is reduced below the level needed to power a systematic energy-requiring destruction of cellular contents from within the dying cell. When cell stress is overwhelming and energy metabolism has been essentially shut down, the cell dies instead by necrosis with the resulting negative consequences for surrounding cells of a tissue. Mitochondria have been called the central executioners in apoptosis because so many of the signals that lead to this form of cell death either originate in mitochondria or are amplified by mitochondrial events. The now classic picture is of mitochondria responding to a cell death signal by disruption of the organelle's outer membrane and release of a set of apoptotic factors from within the intermembrane space including cytochrome c, AIF, procaspase 3, SMAC/DIABLO and several others.

At least two pathways of apoptosis exist, one is dependent on caspases and involves the interaction of released cytochrome c into the so -called apoptosome which initiates the caspase cascade as a key step. The second pathway is caspase-independent and involves the relocation of AIF from the mitochondrion to the nucleus and cytosol.

Many drugs induce apoptosis as an unwanted effect through reactions that alter energy metabolism and other cellular processes. As cancer can be viewed as a failure of cells to undergo timely apoptosis, compounds that induce apoptosis are being sought as a valuable therapeutic.

Assay Details
The ApoTox™ assays are cell-based and use the stepwise release of cellular compounds into the cell medium to serially fractionate cellular compartments for subsequent analysis. It involves a carefully worked-out set of detergent additions that first break the plasma membrane without affecting the mitochondrial outer membrane, followed by a second detergent addition to release the mitochondrial contents, leaving the nuclear pellet untouched. The result is a collection of 3 fractions, which can then be compared by ELISA or by Western blotting to follow the distribution of proteins involved in the apoptotic process. The advantages of the method are speed, the simplicity of the separation, which can be done using a bench top centrifuge (while approaches that separate mitochondria from cytosol physically require an ultracentifuge), and the ability to do many samples simultaneously e.g. a 24 or 96 well plate.

When many compounds are to be screened or for initial determination of and IC₅₀, cytochrome c distribution between the first and second supernatants can be done rapidly using our 96-well plate ELISA assay.

Once it has been established that a compound induces apoptosis and the concentration range identified, a more detailed and quantitative analysis is possible by using Western blotting to locate multiple proteins involved in the process (as shown in figure 1 below). We provide a monoclonal antibody cocktail containing an anti-cytochrome c mAb along with ones to follow a cytosolic component (GAPDH), a protein of the mitochondrial matrix space (PDH) as well as the inner membrane (F1α subunit). With this set it is possible to quantitate the cytochrome c release by controlling for inadvertent mechanical disruption of the organelle.

An additional advantage of the Western blotting protocol is the possibility of expanding the analysis to include additional apoptotic proteins in experiments, for example AIF, SMAC/Diablo and BAX, for a more definitive picture of the apoptotic events ongoing. Figure 2 below shows data for Bax, which, as seen, migrates from the cytosol to the mitochondrial fraction on and/or as there is initiation of apoptosis.

Figure 2. The redistribution of BAX in response to initiation of apoptosis by MPP+ (1-methyl-4-phenylpyridinium) and troglitazone. In both cases there is major redistribution of BAX to the mitochondrion.