Apoptosis means that when cells grow to a certain stage and some physiological processes, they will be strictly regulated by many factors and mechanisms and enter into death. This is a dynamic process, involving a series of complex biochemical reactions, is a cascade reaction involving enzymes, but also involves the expression and regulation of different genes, signal transduction. Several physiological processes can be used to detect apoptosis, so multi-parameter analysis is very important for accurate detection of this process. At the same time, understanding the mechanism of cell death and survival is also a key content in toxicological analysis and drug development and application.
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The MTT assay is a colorimetric assay for assessing cellular mitochondrial activity which correlates with the number of living cells. The enzymes in mitochondria can reduce the yellow soluble MTT into an insoluble formazan with purple color. By measuring the absorbance between 500-600 nm, you can easily estimate the percentage of drug-induced apoptosis based on the viable cell calibration curve. Besides MTT, a series of alternates such as XTT (2,3-bis-(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide), MTS (3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and WST-8 (2-(2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) can be provided by our chemical department and applied in the cell apoptosis assay.
For more precise and reliable detection of cell apoptosis, we offer the service of flow cytometry apoptosis detection (FCAD) assay which can study large number cells individually and show you more morphological information about the cell death. By using our highly specific fluorescent markers, FACD can separate apoptosis from necrosis easily, give the exact number of apoptosis cells, and provide the assessment of early or late stages of apoptosis.
Caspase (cysteinyl-aspartic acid proteases) is a family of proteases, and its activation is a typical feature of apoptosis. Once a caspase enzyme is stimulated, it activates a series of other caspase enzymes, cleaves the corresponding plasma nuclear substrate, and eventually leads to apoptosis. Caspase 3 activation occurs in the early stage of apoptosis and plays an important role in the process of apoptosis. Caspase 3 Activity Assay (CASP3C) can specifically quantify caspase 3 in vitro based on fluorescence immunoenzyme assay.
In the early stage of apoptosis, the phosphatidylserine (PS) located on the inside of the cell membrane migrated to the outside of the cell membrane. Phospholipid binding protein V (Annexin V) is a calcium-dependent phospholipid binding protein, which has a high binding ability to PS. Therefore, Annexin V can be used as a probe to detect phosphatidylserine exposed to extracellular assay. Therefore, using Annexin V, which has a high affinity for PS, Annexin V was labeled with fluorescein (such as fluorescein isothiocyanate FITC),) combined with PI exclusion method (because the necrotic cell PS was also exposed to the cell membrane. The apoptotic cells could be detected by flow cytometry after high staining of PI).
DNA degradation is also one of the typical markers of apoptosis. Its degradation products can be divided into two kinds, namely, high molecular weight single chain DNA gap and low molecular weight DNA fragment (single chain and oligomeric nucleosome). There is a specific method to detect the single-strand DNA gap, that is, enzyme-catalyzed labeling of specific nucleic acids to the free 3'-OH terminal, this method is called TUNEL.
The changes of cell morphology, such as membrane wrinkle or vesicle, nuclear chromatin density, cytoplasmic concentration and so on, can be used as evidence of apoptosis. The progress of apoptosis is generally judged by the morphological changes of nuclear chromatin. The most commonly used DNA specific dyes are Hoechst 33342 (B2261), Hoechst 33258 (B2883), DAPI (D9542). The binding of the three dyes to DNA is non-embedded, mainly in the A / T base region of DNA. Bright blue fluorescence is emitted when excited by ultraviolet light.
Why Choose BOC Sciences?
BOC Sciences is the leading industry in chemical molecules, cell lines culturing and flow cytometry technologies application, providing package services for systemic experimental design, operation and data analysis in cell apoptosis assay. Our scientists team with over a decade of experience in cancer research and apoptotic biology will also give you key tips to avoid common pitfalls and help you choose the right assay for drug discovery.