BOC Sciences provides a variety of apoptosis detection services, involving early, mid and late detection of apoptosis.
Understanding the mechanism of cell death and survival is the key content of toxicological analysis and drug development and application. Since the cell death pathway is complex and dynamic, multi-parameter analysis is very important for the accurate detection of this process.
Figure 1: Graphic representation of apoptosis signaling pathways (Mahmoud Iranpour. 2016).
BOC Sciences offers a wide range of apoptosis assays to measure multiple components on selectable testing platforms to accelerate your research.
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 a large number of cells individually and show you more morphological information about 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. BOC Sciences can specifically quantify caspase 3 in vitro based on fluorescent immunoenzyme assay.
In the early stage of apoptosis, the phosphatidylserine (PS) located on the inside of the cell membrane migrates 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. Annexin V has a high affinity for PS and can be 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 fragmentation and nuclear changes are the characteristics of late apoptosis, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, TUNEL detection, or Hoechst staining. We provide a fast and reliable detection method for studying in situ apoptotic DNA fragmentation.
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. We use DNA-specific dyes for detection and observation, including Hoechst 33342 (B2261), Hoechst 33258 (B2883), and DAPI (D9542), which are non-intercalated with the A/T base region of DNA and emit bright blue fluorescence light when excited by ultraviolet light.
BOC Sciences is a leading service provider in providing chemical molecules, cell line development, and flow cytometry technologies, providing bundled services for systemic experimental design, operation and data analysis in cell apoptosis assay. Our team of scientists 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.
We offer several advanced methods for detecting apoptosis, including MTT assay, Flow Cytometry Apoptosis Detection (FCAD), caspase activity detection, membrane change detection with Annexin V, DNA fragmentation analysis, and cell morphology observation using specific DNA stains.
The MTT assay measures mitochondrial activity, which correlates with viable cell numbers. When cells undergo apoptosis, mitochondrial function declines, and the MTT reagent is reduced to an insoluble formazan, which can be quantified to assess apoptosis levels in your sample.
FCAD is a powerful technique that uses fluorescent markers to distinguish apoptotic cells from necrotic cells. It provides precise, multi-parametric data on the stages of apoptosis, including early and late stages, by analyzing individual cells and their morphological changes.
Caspases are proteases activated early in apoptosis and are crucial for the apoptotic cascade. We measure caspase 3 activity as it plays a central role in initiating the apoptotic process, providing valuable insights into the apoptotic mechanism.
During apoptosis, phosphatidylserine (PS) translocates from the inner to the outer leaflet of the cell membrane. We use Annexin V, a phospholipid-binding protein, to detect exposed PS and differentiate apoptotic cells from necrotic ones using flow cytometry combined with propidium iodide (PI) staining.
DNA fragmentation is a hallmark of late apoptosis. We use techniques like flow cytometry (sub-G1 DNA content), TUNEL, or Hoechst staining to detect and quantify DNA breaks. This provides reliable data on the progression of apoptosis at the cellular level.
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