Apoptosis is a highly regulated mechanism of cell death that is essential to ensure normal development and homeostasis of all multicellular organisms. It counterbalances cell proliferation and differentiation pathways and removes unwanted cells.
An excess level of apoptosis is a factor in neurodegenerative diseases and autoimmune disorders, while failure of apoptosis to destroy aberrant cells is a key element of cancer.
We offer a variety of tools that will enable you to easily and reproducibly measure apoptosis markers such as:
A universal phenomenon during cellular apoptosis is the loss of phospholipid asymmetry in the cell membrane. This results in the exposure of PS on the cell membrane surface. Annexin V binds to PS exposed on the cell membrane, enabling quantification of apoptotic cells by staining them with fluorochrome-conjugated Annexin V.
Annexin V binds to exposed PS irreversibly. Cellular conditions such as physiological stress or inflammation can lead to PS exposure but not necessarily to cell death. To address this problem, we also offer a reversible Annexin probe that can detach from PS when the molecule flips back to the inner membrane.
This reversible Annexin probe will allow you to investigate apoptosis in live cells over time. To further distinguish apoptosis from necrosis, cells can be stained with nuclear dyes such as propidium iodide (PI) that are excluded from entering viable and apoptotic cells.
Caspase assay kits
The caspase family of highly conserved cysteine proteases plays an essential role in apoptosis. Initially synthesized as inactive pro-caspases, caspases become rapidly cleaved and activated in response to granzyme B, death receptors and apoptosome stimuli.
We offer a range of colorimetric/fluorometric caspase assay kits to measure activity of individual or multiple caspases, as well as specific caspase inhibitor drug screening kits:
Calpain and cathepsin assay kits
The importance of caspases in triggering apoptosis is well established. However, inhibition of caspases only results in a delay in apoptosis and not a complete block, highlighting the role of non-caspase proteases such as calpains and cathepsins.
Mitochondrial transmembrane potential is an important parameter of mitochondrial function that is used as an indicator of cell death. The collapse of mitochondrial transmembrane potential coincides with the opening of mitochondrial permeability transition pores, leading to the release of cytochrome C in the cytosol. This, in turn, triggers other downstream events in the apoptotic cascade.
We offer a variety of dyes including JC-1, JC-10 and TMRE to measure mitochondrial transmembrane potential changes. These dyes can be used in your detection platform of choice.
Cell membrane blebbing and chromatin condensation are prominent morphological features of apoptosis. During apoptosis, the cell cytoskeleton breaks up, causing some parts of the cell membrane to bulge outwards. The bulges will eventually separate from the cell, taking a portion of the cytoplasm with them. This forms what are known as apoptotic bodies.
Concurrently, the chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert, highly condensed form that is subsequently fragmented and packaged into the apoptotic bodies.
Together with chromatin condensation, genomic DNA fragmentation is considered a hallmark of the terminal stages of apoptosis. Activation of CAD (caspase-activated DNase) by the caspase cascade leads to specific cleavage of the DNA at the internucleosomal linker sites between the nucleosomes, generating fragments of ~200 base pairs.
These so called DNA ladders can be visualized by agarose gel electrophoresis. Alternatively, DNA fragmentation, especially the presence of DNA nicks, can be shown by terminal deoxynucleotidyl transferase (TUNEL) assays.