Astronomers have finally decided that Pluto is not a planet—76 years after its initial discovery. Fortunately, cell biologists can settle matters much faster. In fact, the latest generation of reagents and assays ensure that your observations provide solid evidence that allow you to draw quick and irrefutable conclusions.
Such efficiency has been welcomed enthusiastically by those studying apoptosis, which occurs daily with billions of cells that “die an altruistic death in order to secure the functionality of the whole organism,” according to a paper published in the August 2005 issue of Cell Death and Differentiation. “Death like this is essential for the regulation of development, and later on for maintenance of tissue homeostasis, being a consequence of a balanced cell death versus cell proliferation ratio.” Disruption or dysregulation in this balance contribute “to approximately half of the medical illnesses,” according to the paper.
Pinpointing how each of these diseases develops means sorting through a complex network of genes, proteins, enzymes, receptors, and signaling molecules. Accomplishing this task is becoming easier as companies introduce kits and assays designed for studying specific components of the apoptosis cascade. Some of these products provide the components to study the biochemical reactions related to apoptosis. Or, you can purchase products that allow you to actually observe the morphological features of apoptosis: nuclear condensation, chromosomal fragmentation, membrane blebbing, cellular fragmentation into membrane-bound bodies, and phagocytosis of the dying cell.
Many researchers use the activity of Cytosolic Aspartate-Specific Proteases (or CASPases), which break down the cell, to gauge the occurrence of apoptosis. You can also assay for other components in the apoptotic cascade that lead up to the activation of caspases. These include an increase of nitric oxide, the exposure of phosphatidylserine on the cell membrane, and the release of cytochrome c from the mitochondria. To assess these components, many of the kits harness fluorescence signaling as indicators. You can evaluate caspase activity by measuring the fluorescence intensity that results from artificial caspase substrates, which give off fluorescent signals after interacting with caspase. You can also find caspase inhibitors that fluoresce once bound to caspase. Best of all, the selection includes kits that are designed for high-throughput screening, high content screening, and flow cytometry.
However, some traditional techniques can be equally powerful. For immunohistochemical staining, you can use a variety of stains to detect apoptosis. One favored technique is measuring the level of beta-galactosidase; higher levels indicate that cells are undergoing apoptosis and not becoming neoplastic. Or, you can use gel electrophoresis to determine the presence of DNA fragments that are characteristic of apoptosis.
These methods, and those listed below, will lead you straight into the orbit of apoptosis. Just peer into the microscope and enter the galaxy of cell death.
Get the information you need for apoptosis detection, all in one place. Invitrogen offers a wide selection of apoptosis products, including antibodies and functional assays, key sections of iProtocols for both flow cytometry and microscopy, as well as free access to Howard Shapiro's Practical Flow Cytometry, Fourth Edition.
Apoptosis Detection Assays
Sigma-Aldrich is pleased to offer a histochemical staining assay to detect the presence of senescent cells. The Senescent Cells Staining Kit (Cat. No. CS0030) identifies senescent cells using a rapid staining procedure. Sigma-Aldrich offers a broad range of cell fractionation and enzymatic assays for apoptosis detection, including mitochondrial staining assays, and annexin, caspase, cathepsin and citrate synthase enzymatic assays. View our apoptosis detection assays now.
The Cell Health Profiling BioApplication is an automated image analysis algorithm that provides detailed analysis of phenotypes related to cell health. This flexible tool allows you to analyze different features of interest, then create an activity profile based on patterns of features. These patterns, termed “Events”, can be defined to detect and distinguish: early and late cytotoxicity, apoptosis and necrosis and survival of target-expressing cells vs. non-expressors. Cellomics’ Cell Health Profiling BioApplication saves you the time and effort required to determine toxic mechanisms of compounds.
No more Annexin V
Upstate® Anti-Phosphatidylserine, Alexa Fluor® 488 from Millipore, is a superior monoclonal antibody that is sensitive and specific for apoptosis detection in flow cytometry and other immunofluorescent applications. Why rely on a recombinant protein when you can have the reliability of a monoclonal antibody? Phosphatidylserine (PS) is a membrane phospholipid that is localized to the inner surface of cell membranes. During apoptosis, PS redistributes from the inner membrane leaflet to both the inner and outer membrane leaflets. “Loss of membrane asymmetry” is a key marker for apoptosis.
