Apoptosis

 Apoptosis is a process of programmed cell death (PCD), it occurs as a cell is damaged beyond repair resulting from cell-infected virus and stressful conditions. This process exhibits compaction of many conditions, including intracellular homeostasis, free radical production, membrane asysmetry, mitochondria function, caspase activation, chromatin condensation, DNA fragmentation, and plasma membrane integrity. Incorrectly regulated apoptosis is implicated in a number of disease states, including cancer, stroke, Alzheimer’s disease, and several autoimmune diseases. Thus, the study of apoptosis pathway can be used to understand and rescue human’s life from the diseases (Ref: http://en.wikipedia.org/wiki/Programmed_cell_death).

 At the present time, cell-based assays have been developed to detect the apoptotic cells and viable cells. For these reasons, Invitrogen offers a diverse selection of assays for apoptosis and cell viability. Many of these assays are fluorescence based; these assays are generally less hazardous and less expensive than radioisotopic techniques, more sensitive than colorimetric methods, and more convenient than animal testing methods. Moreover, the assays can be analyzed on a cell-by-cell basis and some are equally suitable for detection by microscopy, by flow cytometry, or with a microplate reader, and in most cases are suitable for high-throughput applications
(Ref: http://www.invitrogen.com).

Intracellular homeostasis

Intracellular homeostasis of apoptotic cell shows the changes of pH, Ca2+, and Zn2+. The intracellular acidification has been shown to result from a change in the set point of the Na+/H+-antiport (Göbel et al., 2008). The pH sensitive dyes are used to detect acidification in apoptosis. Invitrogen can supply the pH sensitive dyes BCECF AM
(more info: http://probes.invitrogen.com/media/pis/mp01150.pdf)
and caboxy SNARF®-1 Am acetate
(more info: http://probes.invitrogen.com/media/pis/mp01270.pdf) that are compatible with fluorescence microscopy, flow cytometry, and fluorescence spectroscopy. The acetoxymethyl (AM) ester derivative of BCECF is membrane-permeant and has 4-5 negative charges at pH 7.8, promoting noninvasive bulk loading of cell suspensions and good dye retention. The pKa of 7.0 positions BCECF AM within the normal range of cytoplasmic pH (6-8), and its pH-dependent fluorescence excitation profile allows ratiometric measurements to be taken.
The intracellular of Ca2+ concentration have been shown to trigger apoptosis
(ref: www.invitrogen.com).

The acetoxymethyl esters of Ca2+-responsive dyes are cell-permeant, making them useful for staining live cells. Fluo-4 AM efficiently excited at 488 nm exhibits a fluorescence enhancement of at least 100-fold upon binding to Ca2+. The Fluo-4 has higher signals and more sensitive when it is used with FLIPR® system from Molecular Devices. The Fluo-4-NW (No Wash) Calcium Assay is the next-generation calcium indicator from the industry-leading fluo calcium indicator product family. The Fluo-4-NW assay provides superior performance without a quencher dye, combined with the convenience of a no-wash format. This new approach to calcium detection minimizes the potential of nonspecific assay interference and reduces %CV, thereby improving data quality using adherent and suspension cell formats. Fura-2 AM is a UV light-excitable Ca2+ indicator and I the dye of choice for ratiometric Ca2+ measurements by microscopy. An excitation ratio of 340/380 nm is typically used for fura-2 AM; the Ca2+-dependent absorption shift is seen by monitoring the emission at about 510 nm (more info:  http://probes.invitrogen.com/media/pis/mp01240.pdf )

Intracellular Zn2+ concentration is monitored by using RhodZin™-3 AM
(more info: http://probes.invitrogen.com/media/pis/mp07990.pdf), a novel fluorescent Zn2+ indicator for live cells, has nanomolar affinity for Zn2+ , but dose not exhibit any sensitivity to Ca2+ at physiological concentrations. RhodZin™-3 AM localizes to mitochondria and exhibits a large pH-independen florescence increase when bound to intramitochondrial free Zn2+. The detection of mitochondrial Zn2+ is significant, a distinctive feature of the early stages of PCD is the disruption of active mitochondria.

Free Radical Production

 Following an apoptosis signal, cells sustain progressive lipid peroxidation. The fatty acid cis-parinaric acid is a structural analog of intrinsic membrane fatty acids and possesses a very large fluorescence Stodes shift (about 100 nm) and an almost complete lack of fluorescence in water. Oxidation of cis-parinaric acid by lipid hydroperoxides results in a quenching of fluorescence.
 One progressive signal of apoptosis is intracellular reactive oxygen species production. H2DCFDA and CM-H2DCFDA are both cell permeant and nonfluorescent until oxidized in the cytoplasm of live cells. After entering live cells, the diacetate groups are cleaved by intracellular esterases. Oxidation of the reduced dyes can then occur in the presence of ROS, causing the dyes to fluoresce. CM-H2DCFDA is typically better retained by the cells because its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols.
 The image-iT LIVE Green Reactive Oxygen Species (ROS) Detection Kit
(more info: http://probes.invitrogen.com/media/pis/mp36007.pdf) provides the key reagents necessary for the detection of ROS in live cells. The assay provides carboxy-H2DCFDA; the common inducer of ROS production, tert-butyl hydroperoxide (TBHP), as a positive control; and the blue-fluorescent, cell-permeant nucleic acid stain Hoechat 33342. Using this combination of dyes according to the optimized protocol provided, oxidatively stressed and non-stressed cells are reliably distinguished by fluorescence microscopy. 
 MitoSOX™ Red (moreinfo:http://probes.invitrogen.com/media/pis/mp36008.pdf),      mitochondrial superoxide indicator is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells. It is live-cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondrea, MitoSOX™ Red reagent is oxidized by superoxide and exhibits bright red fluorescence upon binding to nucleic acids. MitoSOX™ Red reagent is readily oxidized by superoxide but not by other ROS-or reactive nitrogen species (RNS)-generation systems, and oxidation of the probe is prevented by superoxide dismutase.

Membrane Asymmetry

 The human vascular anticoagulant annexin V is a 35-36 kDa, Ca2+-dependent phospholipids-binding protein that has a high affinity for phosphatidylserine (PS). In normal viable cells, PS is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, exposing PS to the external cellular environment where it can be detected by annexin V conjugates. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of PS, an indicator of intermediate stages of apoptosis. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained by fluorescent annexin V conjugates, as measured by flow cytometry, is typicall about 100-fold. Annexin V conjugates are very useful for flow cytometry and confocal or epifluorescence microscopy and, like antibody staining, can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells. Invitrogen provides the several versatile product that can discriminate live cells from apoptosis cell.
 Apoptosis is often assessed with fluorescent annexin V conjugates to detect phosphatidylserine on the plasma membrane. However there are some situations, where staining cells with annexin V is not the optimal method for detection apoptosis. These include assays where cells are sensitive to the high calcium concentrations required for annexin V binding, assays where phosphatidylserine detection on adherent cells is adversely affected by trypsinization, and assays where washing of samples is prohibitive. Three monomeric cyanine dyes (PO-PRO™-1, YO-PRO®-1, and TO-PRO®-3) have been shown to penetrate apoptotic cells because of permeability changes associated with the loss in asymmetry of the plasma membrane. these dyes enter apoptotic cells and bind nucleic acids while the cell-impermeant dead cell stain propidium iodide (PI) is excluded. The three dyes have unique excitation wavelengths providing enhanced flexibility in multiplexed assays
(more info: http://probes.invitrogen.com/media/pis/mp03602.pdf ).

 
Mitochondrial Function

 MitoTracker® dyes are membrane potential-dependent probes for staining mitochondria in live cells. The fluorescence signal of MitoTracker® dyes is brighter in active mitochondria than in mitochondria with depolarized membranes, providing a way to identify healthy cells in a population. The staining pattern of MitoTracker® dyes is retained throughout subsequent immunocytochemistry, DNA end-labeling, in situ hybridization, or counterstaining steps. MitoTracker® Red CMXRos exhibits minimal spectral overlap when paired with a green-fluorescent dye, while MitoTracker® Orange CMTMRos is compatible with far-red-fluorescent dyes. MitoTracker® Red CM-H2XRos and MitoTracker® Orange CM-H2XRos are best for experiments requiring the lowest fluorescence background.
 JC-1
(more info: http://probes.invitrogen.com/media/pis/mp34152.pdf) is a cationic dye that exhibits potential-dependent accumulation in mitochondria, as indicated by a fluorescence emission shift from green (about 522 nm) to red (about 590 nm), making it useful for ratiometric measurements. The MitoProbe™ DilC1(5) 
(http://probes.invitrogen.com/media/pis/mp34151.pdf) and green-fluorescent DiOC2(3) carbocyanine dyes
(http://probes.invitrogen.com/media/pis/mp34150.pdf) , repectively, along with a mitochondrial membrane potential disrupter, CCCP, for the study of mitochondrial membrane potential. Cells stained with DilC1(5) can be visualized by flow cytometry with red excitation and far-red emission filters cells stained with DilC1(3) can be visualized by flow cytometry with blue excitation and green and red emission filters. Rhodamine 123 and dihydrorhodamine 123 are readily sequestered by active mitochondria without inducing cytotoxic effects.

Uptake and equilibration of these rhodamine dyes is rapid-a few minutes-compared to other membene potential-sensitive dyes, which may take 30 minutes or longer. The methyl or ethyl esters of tetramthylrhodamine (TMRM and TMRE, respectively) are effectively used in multicolour applications that incorporate green-fluorescent dyes.
 The mitochondrial permeability transition pore, a nonspecific channel formed by components from the inner and outer mitochondrial membranes, appears to be involved in the release of mitochondrial components during apoptotic and necrotic cell death.

The Image-iT® LIVE (http://probes.invitrogen.com/media/pis/mp35103.pdf) and MitoProbe™ Transition Pore Assay Kits (http://probes.invitrogen.com/media/pis/mp34153.pdf) provide a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. These assays employ the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl2, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochodria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, whichdoes not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl2, while the fluorescence from the mitochondrial calcein is maintained. The Image-iT® LIVE Mitochondrial Transition Pore Assay Kit also contains MitoTracker® Red CMXRos as a mitochondrial counterstain for imaging.

One trigger for the execution of apoptosis is the release of cytochrome c from the mitochondria. Because cytochrome c is released from mitochondria into the cytosol during apoptosis, its localization can serve as a marker of apoptosis. The SlectFx® Alexa Fluor® 488 Cytochrome c Apoptosis Detection Kit (http://probes.invitrogen.com/media/pis/mp35115.pdf) employs an anti-cytochrome c antibody and an Alexa Fluor® 488 dye-labeled secondary antibody to detect cytochrome c.
 
Caspase Activation

 Caspases are a family of cysteinyl endoproteases that cleave after Asp residues. Evidence clearly links members in the Ced-3 subclass to the initiation and propagation of apoptosis (including such members as caspases-2, -3, 6, -7, 8, -9, and -10), whereas members within the caspase-1 subfamily (such as caspases-1, -4, and -5) are probably involved in proteolytic activation, such as those previously observed for IL-1 or interleukin-18 (Miller, 2002).
 Live cell caspase assays, the Vybrant® FAM Assay Kit, employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology essentially an affinity label. The reaent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amino acid sequence. A carboxyfluorescein group (FAM) is attached as a reporter. The FLICA™ reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA™ molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.
 Fluorogenic caspase substrates are based on specific caspase cleavage sequences that are linked to fluorescent molecules. Cleavage of the substrates changes the fluorescent characteristics of the molecules that are easily detected using fluorescent plate readers and fluorometers, AMC-derived substrates yield the blue-fluorescent product AMC, which has excitation/emission maxima of 342/441 nm upon proteolytic cleavage. Rhodamine 110-based caspase substrates are bisamide derivatives of the fluorophore; peptides are covalently linked to each of the amino groups of rhodamine 110, thereby suppressing both the visible absoption and flurescence of the dye. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted in a tow-step process, first to the fluorescent monoamide and then to the even more fluorescent R110. the R110 cleavage product has spectral properties similar to those of flluorescein and a large extinction coefficient, thus providing ease of use under standard fluorescein filter setups and excellent signal-to-background rations. AFC-based caspase substrates are composed of the fluorphore AFC (7-amino-4-trifluoroethyl coumarin) and a synthetic tetrapeptide. Upon cleavage of the substrate by caspase-3 or related caspases,. Free AFC emits a yellow-green fluorescence. AMC-derived substrate yields the blue-gluorescent product AMC upon proteolytic cleavage.
 The colorimetric protease assay kits provide a simple, convenient method for assaying the activity of specific active caspases. The substrates are composed of a chromophore, p-nitroanilide (pNA), linked to a synthetic tetrapeptide. Upon cleavage of the substrate by a specific active caspase, free pNA light absorbance can be quantified using a spectrophotometer or a microplate reader at 400 or 405 nm. Composition of the absorbance of pNA from apoptotic sample with an uninduced control allows determination of the increase in caspase activity.
 Poly (ADP-ribose) polymerase (PARP) is a 116 kDa nuclear protein which is strongly activated by DNA strand breaks. During apoptosis, ICE family members such as caspase-3 and -7 cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis.
The Anti-PARP FITC Apoptosis Kit (http://tools.stage.invitrogen.com/content/sfs/manuals/AHM2011%20pr937%20revA5%20jun1608%20(PARP%20FITC).pdf) contains a FITC-conjugated anti-PARP antibody that specifically recognizes the 85 kDa (p85) fragment of cleaved PARP. In this technique, apoptosis is detected by flow cytometry using an intracellular staining method. The FITC-conjugated antibody specifically binds to the cleaved PARP, not the full-length intact PARP protein. Cells are then analyzed by flow cytometry, and positive FITC staining is a measure of the percentage of cells undergoing apoptosis. Moreover, the BioSource™ Cleaved PARP [214/215] ELISA (http://tools.stage.invitrogen.com/content/sfs/manuals/KHO0741%20pr360%20revA3%20jun1608%20(Cleaved%20PARP%20[214-215]).pdf)  is designed to detect and quantify the levels of the PARP p85 fragment, which is produced from full-length PARP by cleavage between Asp214 and Gly215 during apoptosis. This assay is intended to detect cleaved PARP [214/215] from human cell lysates or tissue homogenates.

Chromatin Condensation and DNA fragmentation

Cell undergoing apoptosis display an increase in nuclear chromatin condensation; these compact nuclei become hyperfluorescent when labeled with some nuclear stains. Hoechst 33342 (http://probes.invitrogen.com/media/pis/mp21486.pdf) is a UV-excitable nucleic acid stain readily taken up by all cells. Its blue fluorescence is particularly bright in the condensed nuclei of apoptotic cells. By contrast, propidium iodide only enters cells with compromised plasma membranes. The staining pattern resulting from the simultaneous use of these two nucleic acid stains makes it possibleto distinguish healthy, apoptotic, and dead cell population by flow cytometry or fluorescence microscopy.
DNA strand breaks that occur during apoptosis are often identified using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay, which employs a terminal deoxynucleotidyl transferase (TdT) to add BrdUTP to the fragmented DNA ends. The BrdU is then detected with an anti-BrdU antibody. For convenience, the APO-BrdU™ TUNEL Assay Kit (http://probes.invitrogen.com/media/pis/mp23210.pdf ) includes buffers and reagents to carry out the BrdU incorporation, an Alexa Fluor® 488 dye-labeled anti-BrdU antibody, and samples of fixed cells that serve as both positive and negative controls. To directly label DNA strand breaks, the ChromaTide® BODIPY® FL-14-dUTP (http://probes.invitrogen.com/media/pis/mp07603.pdf) and ChromaTide® Texas Red®-12-dUTP (http://probes.invitrogen.com/media/pis/mp07603.pdf) analogs can be added to DNA strand breaks in a standard terminal deoxynucleotidyl transferase reaction. For the incorporation and subsequent identification of BdrU, BrdUTP and conjugates of an anti-BrdU mouse IgG1 monoclonal antibody are available in a range of fluorescent colors to suit your application.
In vitro DNA fragmentation detection can use SYBR® Green I nucleic acid stain is an exceptionally sensitive nucleic acid gel stain that has bright fluorescence when bound to dsDNA and low background in gels, making it a good choice for nucleic acid staining in vitro. SYBR® Green I dye reveals laddering in bulk preparations of DNA and detects apoptotic DNA fragmentation in single cells using the comet assay. The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit (http://tools.stage.invitrogen.com/content/sfs/manuals/BioSource%20KHO1021.pdf) provides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other kit protocols that require 1 to 2 days to finish, this detection method requires only less than 90 minutes to prepare DNA in a single tube without the need for extraction or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis.

Plasma Membrane Integrity

 As cells progress further down the apoptosis pathway, breakdown of the plasma membrane allows nucleic acid stains that are normally impermeant to an intact plasma membrane to pass through the cell to interact with nucleic acids.
 SYTOX® Nucleic Acid Stains (http://www.stage.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/SYTOX-Dead-Cell-Stains.html) are high-affinity nucleic acid stains that easily penetrate cells with compromised plasma membranes and yet will not cross the membranes of live cells. Each of the four SYTOX® Nucleic Acid Stains is excited by a unique laser, providing ultimate flexibility in designing multicolor experiments.

Muliparametric Analysis

 Since there is no single parameter that defines PCD, a combination of techniques is recommended for reliable detection of apoptosis. Vybrant® Apoptosis Assay Kits (more info: http://www.stage.invitrogen.com/site/us/en/home/Products-and-Services)   are designed to selectively differentiate apoptotic cells from living and necrotic cells in a single cell population. The kits were designed to detect various apoptotic parameters in a single flow cytometry assay, helping to dissect the early and middle stages of the apoptosis response.

For more information, you can go to www.invitrogen.com .