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Apoptosis

Apoptosis is also known as programmed cell death. The morphologic and biochemist of apoptosis is distinct from the cell death. The characteristic morphological signs of apoptosis are blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay.

Apoptosis is a highly organized process of programmed cell death for removing unwanted cells from the body during organ development, tissue remodeling, and immune responses. Defects in apoptosis pathways are implicated in the development of cancer and have been extensively linked to the resistance of tumors to chemotherapy. Therefore, an increasing number of anticancer drugs are designed to target specific aberrant signaling components of cell death or survival pathways, including small molecule inhibitors and protein therapeutics.

Figure 1. A simplified view of cell death pathways and therapeutic targets for cancer therapy. (Brunelle, 2010)

Apoptosis Assays at Creative Animodel
Many novel drugs are designed to activate one or more cell death pathways in the target cells. Therefore, their bioactivity can be measured in theory by assessing any critical event within the cell death pathway. At Creative Animodel, our apoptosis assays include an early upstream signal transduction event, such as receptor phosphorylation, or a late cellular response, such as proliferation or cell death.Annexin V-FITC/PI Staining
In early apoptosis, phosphatidylserine (PS) residues, which is positioned inside the plasma membrane, exposed to the cells outer surface. Annexin V has high affinity for PS. Meanwhile, cell membrane in early apoptosis doesn't allow DNA binding dyes to get through. So apoptotic cells can be detected by flow cytometry using fluorescent labeled Annexin V and PI antibody. This approach for apoptosis analyzing provides early, intermediate, and late apoptotic stages simultaneously.
Cell Viability / Cytotoxicity Assays
Cell viability / cytotoxicity assays are the major category of evaluating the activity of an anticancer drug. They are designed to measure either the number of live cells or the number of dead cells in a test sample. In general, these assays involve measuring dye uptake or the release of cytoplasmic enzymes through damaged cellular membranes.

Figure 2. Illustration of cell viability and cytotoxicity assays.

Cell viability assays measure the number of live cells utilizing an enzymatic reaction occurring inside the living cells (e.g. MTT assay). The dead cells can be detected by dye uptake (e.g. propidium iodide, PI) through damaged plasma membrane. Hoechst stain detects condensed nuclei which can be counted using microscopy. Fluorescent Caspase Substrates Analysis
Some novel drugs are intended to kill cancer cells by inducing apoptosis, which is characterized by activation of a caspase cascade. Thus, their bioactivity can be assessed by measuring the caspase activity in the treated cells. Caspases recognize a four amino acid sequence of their substrate, of which the aspartic acid residue is the cleavage site. The available kits provide a substrate (e.g. AMC, AFC, pNA, or rhodamine 110) that becomes fluorescent upon caspase cleavage.
Western Blot
Caspases are a family of protease enzymes playing essential roles in programmed cell death (including apoptosis, pyroptosis, and necroptosis) and inflammation. Caspase activity can be measured by Western blotting analysis using antibodies recognizing cleaved caspase fragments.

The outcome which includes, but not limited to:Flow cytometry results contain data analysis and related imagesThe ratio of dead cells and viable cells, and light microscopy picturesGrayscale analysis of western blot, and exposure results

Creative Animodel can provide high-quality apoptosis services. We promise to offer services with the most competitive price and reliable data to our clients. Please don't hesitate to contact us for more information.

Reference:
Brunelle, J. K.; et al. Apoptosis assays for quantifying the bioactivity of anticancer drug products. Drug Resistance Updates. 2010, 13(6), 172-179.

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