Apoptosis Assays and Probes

Apoptosis

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Cells of multicellular organisms are highly organized and closely regulated to ensure a proper homeostatic balance is maintained so that the organism may function optimally. Cells are typically regulated in two ways: one via the rate of proliferation (cellular division), and the other being the rate of apoptosis (cell death). Cells that are no longer necessary induce suicide by activating intracellular death signaling pathways through a process known as programmed cell death or apoptosis. For example, during frog development, the cells required for tail formation in aquatic tadpoles will initiate apoptosis causing these cells to die. As the tadpole matures into a more terrestrial based adult frog, its tail will disappear entirely.

Mechanism of Apoptosis

Apoptosis occurs normally during development and aging and also as a defense mechanism in response to cellular damage or an immune reaction. It has been linked to normal cell turnover, proper development and functionality of the immune system, hormone-dependent atrophy and chemical-induced cell death. Apoptotic biochemical events lead to specific characteristic or morphological cellular changes essential for a controlled and precise cell death without any damaging or adverse inflammatory responses. These morphological changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal fragmentation.

Apoptosis begins with the cell shrinking and condensing. The cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear DNA breaks up into fragments. These organic cellular fragments generated during apoptosis are referred to as apoptotic bodies. The cell surface is then modified in such a way to express specific surface markers that cause the apoptotic cells to be quickly phagocytized by the immune system before any leakage of its contents occurs. A key advantage of apoptosis is the recycling of apoptotic bodies or organic components of apoptotic cells by the ingesting cells.

Cell Necrosis: A Disparity to Apoptosis

Contrasting to programmed apoptosis, cells and living tissues may experience unprogrammed, premature death as a result from acute cell injury, infection, or inflammation known as cell necrosis. Necrosis impairs the cells homeostatic abilities resulting in an influx of water and extracellular ions causing the cell to swell and burst. The cytoplasmic contents of necrotic cells spill into the extracellular fluid initiating an intense inflammatory response which is extensively damaging to cells and tissue.

Caspase Assays and Probes

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The mechanisms necessary for apoptosis involve highly complex, energy-dependent cascading pathways which are regulated by a family of proteases known as caspases. They are present in all cells as latent enzymes or inactive precursors known as procaspases. Caspase activity is tightly regulated inside a cell to ensure that apoptosis remains inactive until desired. When apoptosis is necessary, caspases initiate activation by proteolytic cleavage. These caspase enzymes are crucial mediators of the complex biochemical events associated with apoptosis. Therefore various colorimetric, fluorimetric and flow cytometric caspase assays have been developed for the detection and examination of active caspases in apoptotic-induced cells.

Caspase assay techniques have been introduced to exam the activity, substrate specificity and the activation status of caspase activity in correspondence to its position in the caspase cascade pathway. Understanding the specific function of each enzyme in the caspase cascade can aid as a determinant in deciding the appropriate assay that will produce the most optimal results. Fluorimetric and colorimetric assays use caspase enzyme activity and specific reporter substrate to generate a detectable signal that can be measured by a microplate reader or imaged microscopically. Cleavage of the substrate is initiated by the interaction between the caspase enzyme and the reporter substrate. Upon cleavage from the substrate, the fluorogenic chromophore will change color or emit a fluorescence which is proportional to the activity of the caspase enzyme present. Fluorimetric assays have a slight advantage when compared to colorimetric assays because of their higher sensitivity, ability to measure across a broader dynamic range, and the tendency to have a lower background signal. The main advantages associated with colorimetric assays are that the signal is detected via a visible color change and it is relatively inexpensive compared to other commercially available assays.

To detect and analyze caspase activity, AAT Bioquest offers a diverse selection of caspase inhibitors, chromogenic and fluorogenic caspase substrates and caspase kits. Our chromogenic caspase substrates are based on the modification of a specific peptide sequence with 4-nitroaniline (4-PNA). Furthermore, we are the only company that offers multicolor substrates of four distinct fluorescence colors based on 7-Amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin (AFC), Rhodamine 110 (R110) and ProRed™. These substrates are available individually as well as packaged together in a kit to simultaneously monitor the activation of multiple caspases in multiplexing assays. (See table below for a quick overview of apoptosis assays and key features)

Caspases: A Family of Proteases

Caspases are cytosolic aspartate-specific, cysteine proteases which serve as the primary mediators of apoptosis. Their activation is regulated by members of the Bcl-2 and IAP protein families. Activation is stimulated upon the receipt of extrinsic or intrinsic death signals which causes intracellular adaptor molecules to aggregate procaspases at receptor-associated cytosolic complexes. Aggregation of procaspases initiates caspases activation via dimerization or dimerization followed by autoproteolytic cleavage. The initial caspase activation causes additional caspases to proteolyze intuitively amplifying the caspase cascade.

Apoptotic Initiator Caspases and Their Respective Assays

Depending upon their role in the caspase cascade, caspases are further subdivided into apoptotic initiator caspases and apoptotic effector caspases. Apoptotic initiator caspases such as caspase-2, -8, -9, and -10 can initiate the caspase activation cascade. For example, caspase-8 is essential for the formation of the death-inducing signaling complex (DISC) and when activated, caspase-8 activates downstream effector caspases. Caspase-8 has proven to show relatively high substrate selectivity to the IETD peptide sequence. AAT Bioquest exploits this enzyme-substrate interaction to develop efficient and robust kits for the detection of caspase-8 activity.

AAT Bioquest's provides three options of its Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit for compatibility with different filters. Each kit contains a unique fluorogenic indicator comprised of a specific fluorogenic chromophore AMC, R110 or ProRed™ conjugated to the substrate selective IETD peptide sequence. This assay can be readily optimized for high throughput screenings and to quantify the activity of activated caspase-8 in apoptotic cells or screen for caspase 8 inhibitors.

Apoptotic Effector Caspases and Their Respective Assays

Apoptotic effector caspases such as caspase-3, -6, and -7 may not be responsible for initiating the cascading pathway but when activated, they play an integral role in the intermediate and later steps of the cascade. For example, caspase-3 is a crucial effector as it amplifies the signal from an initiator caspases and signifies full commitment to cellular disassembly. Detection of activated caspase-3 is carried out using a DEVD peptide sequence which is selective for caspase-3 and proven successful at developing caspase-3 substrates. AAT Bioquest offers two unique set of kits both for assaying caspase-3/7 activity: an Amplite^® Fluorimetric Caspase 3/7 Assay kit and a Cell Meter™ Caspase 3/7 Activity Apoptosis kit.
Amplite^® Fluorimetric Caspase 3/7 Assay kits have three available options. Each kit contains a caspase-3 selective DEVD peptide sequence modified with either the fluorogenic chromophore AMC, R110 or ProRed™, each with its own unique advantage. The Z-DEVD-R110 based caspase substrates exhibit a narrower dynamic range due to their two-step cleavage process. They are more sensitive than coumarin-based caspases substrates and are recommended for end point assays. AMC and AFC–based caspase substrates are best suited for kinetic assays. While our ProRed™-DEVD substrates exhibit longer excitation and emission wavelengths making them extremely useful for screening caspase3/7 inhibitors.

Cell Meter™ Caspase 3/7 Activity Apoptosis kits are also available in three options each with a fluorogenic indicator substrate modified with a different fluorogenic chromophore. These kits are designed to monitor apoptosis through measuring caspase-3 activation. They are robust and can be readily adapted for high throughput assays in a wide variety of fluorescence platforms such as microplate assays.

Signaling Pathways For Apoptosis

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Apoptosis is traditionally considered an irreversible process and therefore the initiation of apoptosis must be tightly regulated by specific activation mechanisms. The two most-widely studied and understood apoptosis activating mechanisms are the extrinsic and intrinsic pathways. AAT Bioquest's caspase assay kits have been optimized to detect and analyze the activation of caspase enzymes as they transmit the apoptotic signal down either their extrinsic or intrinsic pathway.

Extrinsic Pathway

Extrinsic apoptotic signaling pathways responsible for initiating apoptosis use transmembrane receptor meditated interactions between tumor necrosis factor (TNF) receptors and their corresponding ligands or death receptors. TNF receptors are comprised of cysteine-rich extracellular domains and an 80 amino acid cytoplasmic domain. Dimerization of death receptors with its corresponding ligand activates the receptor complex to begin transmittance of the apoptotic signal. Upon dimerization, recruitment of adaptor proteins such as FADD initiates the recruitment of procaspase-8 to the death inducing signaling complex. Oligomerization of the procaspase-8 to the death inducing signaling complex results in the proteolytic cleavage and activation of caspase-8. Caspase-8 triggers the execution of apoptosis in the cell by directly activating other members of the caspase family to initiate the caspase cascade. (For the extrinsic pathway mechanism refer to Image 1)

Caspase binding assays are excellent at detecting key events in the extrinsic signaling pathways responsible for initiating apoptosis such as the activation of initiator and effector caspases. AAT Bioquest offers a comprehensive line of Cell Meter™ Live Cell Caspase Binding kits which use fluorescent cell permeable and nontoxic indicators to detect caspase-1, -2, -3/7, -6, -8, -9, -10 and 13 activities. Upon binding to their appropriate caspases, the fluorescent reagents are retained inside the cell. This binding event impedes any further catalysis of the caspases without halting the apoptosis process. These caspase binding kits are applicable for fluorescence microscopy, flow cytometry and fluorimetric microplate readers. Caspase assays may be performed in conjunction with other apoptosis assays to elucidate any relationship between extrinsic and intrinsic apoptosis pathways. For example, using caspase binding assays in conjunction with assays that detect changes in the membrane potential of mitochondria may reveal any potential "cross-talk" between extrinsic and intrinsic apoptotic signaling pathways.

Intrinsic Pathway

Intrinsic apoptotic signaling pathway is mediated by mitochondrial signaling proteins. It functions in response to various types of intracellular stimuli such as DNA damage, growth factor withdrawal, and death receptor stimulation. Receipt of the intracellular stimulus activates pro-apoptotic proteins such as BH3 interacting-domain death agonist or BID. Other members of the Bcl-2 family such as Bax, interact with BID proteins allowing for their insertion into the outer mitochondrial membrane. The mitochondrial membrane destabilizes allowing for the formation of mitochondrial-apoptosis induced channels, which facilitate the release of cytochrome c into the cytoplasm. Cytochrome c is a pro-apoptotic factor that interacts with the protein apoptotic protease-activating factor-1 (APAF-1) which recruits procaspase-9 to form an apoptosome complex. Formation of this complex cleaves and activates apoptotic initiator caspase-9 to initiate the caspase cascade, ultimately leading to apoptosis. (For the extrinsic pathway mechanism refer to the Image 1 above)

Detecting changes in the mitochondrial membrane potential (MMP) is a great indication that apoptosis has been activated intrinsically. AAT Bioquest has developed proprietary cationic mitochondrial probes sensitive at detecting any changes to the mitochondrial membrane potential of a target cell. These proprietary probes are made available as a key component in AAT Bioquest's Cell Meter™ MMP assay kits for monitoring cell viability. In normal cells, these mitochondrial probes emit a red fluorescence whose signal intensity is proportional to the amount of probes that have accumulated inside the mitochondria. The more probes able to accumulate inside the mitochondria, the more intense the fluorescence signal emitted. However, in apoptotic cells, the fluorescence intensity of the mitochondrial dyes diminishes following the collapse of the MMP indicating the release of cytochrome C into the cytosol which triggers the apoptotic caspase cascade. These kits have been individually optimized for screening apoptosis activators and inhibitors in fluorescence microscopy and for fluorimetric and flow cytometric assays. This kit can be used in conjunction with other reagents, such as Cell Meter™ Phosphatidylserine Apoptosis Assay Kit for multi-parametric studies of cell vitality and apoptosis.

Other Assays For Apoptosis

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A variety of parameters other than detecting changes in mitochondrial membrane potential or activation of caspase enzymes can be used for monitoring cell apoptosis. For instance, decreases in cellular glutathione (GSH) concentration via direct GSH oxidation or GSH export from the cell, has been reported to be an early event in apoptotic signaling. Reduced GSH is a key component in defending against free radicals and for maintaining intracellular redox levels of cells. GSH is freely distributed in the cytosol as well as being compartmentalized in organelles such as the mitochondria. Mitochondrial GSH (mGSH) plays an essential role in repairing and defending against damages caused by oxidative stress and the constant generation of reactive oxygen species (ROS). Studies have shown that the depletion of mGSH will trigger the alternation of mitochondrial permeabilization and the activation of effector caspases. To measure the concentration of reduced GSH, AAT Bioquest has developed a proprietary non-fluorescent Thiolite™ Green dye available in the Cell Meter™ Intracellular GSH Assay Kit. In live non-apoptotic cells, the green fluorescence intensity is increased by the accumulation of Thiolite™ Green in the cytosol and mitochondria of the target cell. In apoptotic and dead cells, the fluorescence intensity of Thiolite™ Green is reduced by the decreased concentration of GSH. Cells stained with Thiolite™ Green can be visualized with a flow cytometer and can be used in conjunction with other apoptosis assays for multi-parametric studies. (Example data analysis of this change in fluorescence intensity using the Cell Meter™ Intracellular GSH Assay Kit can be found below in Figure 1)

Detection of the Early Stages of Apoptosis via Annexin V Conjugates:

Detecting the morphological changes of the cell membrane is an efficient way to detect early stages of the apoptotic process in living cells. The monitoring of these changes, such as in the loss of membrane symmetry, can be accomplished by detecting the expression of the cytosolic phospholipid, phosphatidylserine (PS). During the initial stages of apoptosis, the cell's membrane destabilizes allowing PS to translocate from the cytoplasmic side of the cell to the outer leaflets of the cell membrane where they can be detected via Annexin. Annexin is a family of calcium-dependent phospholipid-binding proteins involved in signal transduction that have a preference for binding PS. AAT Bioquest, has developed a set of comprehensive Annexin V proteins conjugated to our line of iFluor^® dyes. Together they make up PS sensors that specifically bind to PS with excellent photostability. For a more sensitive assay or when dealing with a small or limited sample size, use AAT Bioquest's Cell Meter™ PS apoptosis kits packaged with Apopxin PS sensors. Apopxin's high affinity for PS makes for a more robust assay than other commercial Annexin-V based kits.

The following Figure 2 is an example of detection of binding activity of Annexin V-mFluor Violet™ 450 with PS in Jurkat cell apoptosis using a flow cytometer. In live non-apoptotic cells, Annexin V-mFluor Violet™ 450 detects innate apoptosis in non-induced cells, which is typically 2-6% of all cells. In apoptotic cells Annexin V-mFluor Violet™ 450 binds to PS, which is located on the outer leaflet of the cell membrane, resulted in increased staining intensity.

For the complete line of apoptosis assays, check out AAT Bioquest's comprehensive Cell Apoptosis and Proliferation Catalog online under the Resources Tab. There you can find assays for detecting late stages of the apoptotic process via monitoring DNA fragmentation and condensation, Cell Necrosis assays, Autophagy Assays, and more.

 

References

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