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How to Detect Key Features of Apoptosis?

Cell death plays an important role in homeostasis. It is also a physiological response to various stressful conditions such as infection, oxidative stress, etc. Dysregulation of cell death pathways leads to numerous human diseases. Therefore, a comprehensive understanding of the mechanism would advance our knowledge and aid in development of novel therapeutics. Cell death involves different cellular pathways. One of them is apoptosis, which is a programmed cell death pathway. The apoptotic pathway is highly conserved and involves a complex energy-dependent cascade of molecular events. There are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. However, there is now evidence that the two pathways are linked and that molecules in one pathway can influence the other.

Intrinsic Apoptotic Pathway

Intrinsic apoptosis is triggered by diverse intracellular cell stress signal that results in either caspase dependent or caspase independent cell death cascade. Caspases are a family of cysteine proteases whose functions are linked with apoptosis. Apoptosis is triggered when pro-death signals outweigh pro-survival signals. Apoptotic cell death pathways have three key steps: the induction phase, in which various signals trigger signal transduction pathways. The effector phase, in which multiple stimuli mediated pathways converge and signal cell death and the degradation phase, in which cell structures are destroyed. Once the balance is tipped, cells activate upstream initiator caspase-8 (CASP8) or caspase-9. These caspases cleave and activate executioner caspase-3 and caspase-7. Executioner caspases cleave hundreds of cellular targets, resulting in biochemical and morphological hallmarks and ultimately cell death.

The effector phase of caspase dependent intrinsic apoptosis involve stress signal mediated binding between one of the pro-apoptotic Bcl-2 (B Cell Lymphoma 2) family proteins such as Bak or Bax. Activated Bak and Bax oligomerizes and induce the formation of pores and mitochondrial outer membrane permeabilization (MOMP). Cytochrome c is released from the mitochondria following MOMP and binds to a scaffold protein APAF1 (apoptotic protease-activating factor 1) to form the apoptosome. This event results in activation of procaspase-9 and its subsequent cleavage and activation of caspase-3 leading to the degradation of a series of cellular substrates and subsequently cell death.

Extrinsic Apoptotic Pathway

The extrinsic apoptosis is initiated by perturbations of the extracellular microenvironment that are detected by plasma membrane receptors, propagated by CASP8. This pathway is driven by either of two types of plasma membrane receptors: (1) death receptors (DR), whose activation depends on the binding of specific ligand(s) and (2) dependence receptors, whose activation occurs when the levels of their specific ligand drop below a specific threshold. Death receptors include Fas cell surface death receptor CD95 or APO-1. Death receptor signaling can also lead to NF-κB activation, generally resulting in cell survival associated with a robust inflammatory response.

Methods to Detect Early and Late Apoptosis

Induction of cell apoptosis leads to the exposure of "eat me" signals on the cell surface before the late apoptotic event of cell lysis. Exposure of phosphatidylserine (PS) residues on the extracellular surface of the plasma membrane, has been identified as an early hallmark of early apoptosis. Phospholipid-binding proteins such as Annexin V bind with a high affinity to PS, in the presence of Ca2+. Given that Annexin V is not cell permeable, the binding of externalized PS is selective for early apoptotic cells. Considering that Annexin V is commonly conjugated to fluorescein (FITC), apoptosis detection in green fluorescent protein (GFP)-expressing cells can be problematic. The spectral overlap between the emission profiles of these two fluorophores makes differentiation between signals difficult. GFP CERTIFIED® Apoptosis/Necrosis Detection Kit includes was specifically designed for use with GFP-expressing cell lines and cells expressing blue or cyan fluorescent proteins. The kit includes all the necessary reagents for determination of early and late stages of apoptosis as well as necrosis. An Annexin V-EnzoGold (enhanced Cyanine 3) conjugate enables detection of apoptosis distinct from fluorescein or GFP. The Necrosis Detection Reagent is a red-emitting dye facilitates late apoptosis and necrosis detection.

Figure 1: Distinguishing between healthy, early and late apoptotic and necrotic cells and compatible with green fluorescent probes. GFP CERTIFIED® Apoptosis/Necrosis Detection Kit.

Cytochrome c Release Assay

During apoptosis, cytochrome c is release from the mitochondria into the cytosol and subsequently activating a caspases. This has make cytochrome c release one of several key features use to detect apoptosis. Several kits based on the enzyme-linked immunosorbent assay (ELISA) principle are available to measure cell death related parameters in cell culture supernatants, intact cells, subcellular fractions, tissue extracts or body fluids. Our Cytochrome c (human) ELISA kit has been validated and optimized for quantitative determination of Cytochrome c in with both cytosolic and mitochondrial cell fractions and is supplied with the cell fractionation buffers.]

Figure 2: A typical standard curve. Cytochrome c (human) ELISA kit.

Comet Assay

Hallmarks of apoptosis include cell shrinkage, membrane blebbing, nuclear condensation, and DNA fragmentation. The Comet SCGE assay kit is a fast and simple electrophoresis method for the detection of apoptosis. This method measures DNA damage by fluorescently detecting the integrity of DNA liberated from cells embedded in low melting point agarose. Upon electrophoresis, fragmented DNA of apoptotic cells produces a characteristic "comet" shaped tail as small DNA fragments migrate in the gel more rapidly than in-tact genomic DNA. The length of the tail of DNA from a cell corresponds to the degree of apoptosis.

Figure 3: CaSki cells were treated with 100mM hydrogen peroxide for 20 min at 4°C. Comet SCGE Assay was performed and slides were imaged using a FITC filter. Comet SCGE assay kit.

Caspase Assay

During apoptosis, various caspases are activated, which in turn cleave other proteins within the cell to activate apoptosis. A variety of reagents for caspase activity assays and enzymes are available. Caspase assays are used to distinguish between early or late stages apoptosis. For example, the Caspase 3 cell assay kit can be used to confirm apoptosis in cell extracts by detecting caspase-3 and caspase-3-like (cellular DEVDase) activity, one of the principal caspase activities found in apoptotic cells. Caspase-3 is a member of the interleukin-1 converting enzyme (ICE) family of cysteine proteases. The caspase-3 is activated during apoptotic signaling events by upstream proteases including caspase-6, caspase-8 and cytotoxic T-cell-derived granzyme B. Caspase-3 is one of the principal caspase activities found in apoptotic cells. Targets of caspase-3 cleavage include poly(ADP-ribose) polymerase (PARP) , nuclear lamins, gelsolin and others.

Figure 4: Cleavage of DEVD-pNA by cell extracts and purified caspase-3. Caspase 3 cell assay kit.

Chemiluminescent Cell Viability Assay

Apoptotic cell exhibits a significant decrease in ATP level and loss of ATP levels in cell is an indicator of cell death. In contrast, cell proliferation has been recognized by increased levels of ATP. The ApoSENSOR™ Cell Viability assay kit utilizes bioluminescent detection of the ATP levels for a rapid screening of apoptosis and cell proliferation simultaneously in mammalian cells. The assay utilizes luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer.

Figure 5: ApoSENSOR™ Cell Viability assay kit effectively detects apoptosis. Apoptosis was induced in Jurkat cells and ATP levels were analyzed. Time course showing decrease in ATP levels following induction of apoptosis with camptothecin. The decrease in ATP level correlates with the increase in annexin V positive staining cells.

Chromosome Condensation

Another method for the detection of apoptosis assesses the condensed chromosomes that occur during apoptosis. During apoptosis, the chromosome undergoes goes distinct stages of chromosome condensation. ENZO Life Sciences is a pioneer life sciences company and offers several tools to detect cell deaths. NUCLEAR-ID® Green chromatin condensation detection kit for fluorescence microscopy, flow cytometry and microplate assays. Cell permeable DNA staining dyes fluoresce brighter as the chromosome condenses, differentiating apoptotic cells with condensed nuclei from normal cells.

Figure 6: Chromatin condensation as observed by fluorescence microscopy using a standard 488nm laser. HeLa cells were treated for 4 hours with DMSO (Control) or 2 µM Staurosporine on a slide and stained with 5 µM NUCLEAR-ID® Green dye. The intercalating dye exhibits increased fluorescence upon chromatin condensation, a hallmark of apoptosis. NUCLEAR-ID® Green chromatin condensation detection kit.

Loss of Mitochondrial Membrane Potential Measurement

Biochemical markers of apoptosis include loss of mitochondrial membrane potential. It has been found that compromised membrane potential induced by drug accumulation in the mitochondria contributes to the toxicity of various organs, especially the heart and liver. MITO-ID® Membrane Potential Cytotoxicity Kit measures mitochondrial membrane potential with a cationic dye that fluoresces either green or orange depending upon mitochondrial membrane potential status. In energized cells, the MITO-ID® membrane potential dye exists as a green fluorescent monomer in the cytosol and also accumulates as orange fluorescent aggregates in the mitochondria. However, in cells with compromised mitochondrial membrane potential, the MITO-ID® membrane potential dye exists primarily as green fluorescent monomers throughout the cytosol and no longer exhibits orange fluorescence in the mitochondria.

Figure 7: MITO-ID® Membrane Potential Measurements. Detect mitochondrial perturbations with 10 times more sensitivity than JC-1. Mitochondrial membrane potential (MMP) was evaluated in HeLa cells treated with CCCP using MITO-ID® dye (red) or JC-1 (blue). Using a conventional fluorescence microplate reader, MMP was shown to decrease with increasing CCCP concentration as indicated by a decrease in orange fluorescence. Improved aqueous solubility of the dye and no-wash protocol minimizes variability, leading to a higher Z-factor (> 0.9) than that obtained with JC-1. MITO-ID® Membrane Potential Cytotoxicity Kit.

An overview of Apoptosis Assays offered by Enzo. Complete list of products can be found on the Cell Death page.

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Apoptosis Marker What is being Measured Detection Method Assay / Tool
Phosphotidyl serine detection (PS) Detection of early apoptosis staining of PS on outer cell membrane Fluorometric GFP-CERTIFIED® Apoptosis/Necrosis detection kit
Mitochondrial membrane integrity Measurement of membrane potential Fluorometric MITO-ID® Membrane potential cytotoxicity kit
Caspase activation cascade Measurement of specific caspase activity in cells Colorimetric and Fluorometeric Caspase assay Caspase-3 cell assay kit
DNA fragmentation Measure DNA damage by of DNA fragments migration during gel electrophoresis Fluorescently detection Comet SAGE assay kit
Cytochrome C release ELISA Colorimetric Cytochrome c (h) ELISA kit
Cell viability assay Bioluminescent detection of the ATP level Chemiluminescent ApoSENSOR™ Cell viability assay kit
Chromosome condensation Detects nuclear condensation Fluorometric NUCLEAR-ID® Green chromatin condensation detection kit

Enzo Life Sciences offers a range of products for Cell death including apoptosis, autophagy and related cellular processes. As Scientists Enabling Healthcare™, we realize the value in providing relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to provide simple yet useful tips and guidance for your research needs. Please check out our Successful Research Tips or contact our Technical Support Team for further assistance.

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