By Gil Mor, Ayesha B. Alvero
In Apoptosis and melanoma: tools and Protocols, Second Edition, professional researches within the box aspect the functionality of molecular and mobile biology innovations for learning and detecting the activation of the apoptotic pathway. Chapters specialize in assays constructed to observe its activation not just in vitro but additionally in vivo, optimized multiplex research, medium- to high-throughput displays, and the mobile procedure. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and key pointers on troubleshooting and heading off identified pitfalls.
Authoritative and functional, Apoptosis and melanoma: tools and Protocols, moment variation aids scientists as a stand-alone source for the execution and research of the defined protocols and as a reference for the examine and detection of apoptosis inside and outdoors the realm of melanoma learn.
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Extra info for Apoptosis and Cancer: Methods and Protocols
NVC = events corresponding to viable cells after culture (number of annexin V-negative cells). References 1. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556 2. Russell JH, Dobos CB (1980) Mechanisms of immune lysis. II. CTL-induced nuclear disintegration of the target begins within minutes of cell contact. J Immunol 125:1256–1261 3. Cohen JJ, Duke RC (1984) Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death.
Commonly used methodologies are however performed at a single, user-defined endpoint. We describe a kinetic multiplex assay incorporating the CellPlayerTM NucLight Red reagent to measure proliferation and the CellPlayerTM Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyteTM ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics.
Condensation will form on the plate when transferred from ambient temperature to the 37 °C, humidified environment. Scanning right away will cause the images to appear out of focus. Allow the plate to equilibrate in the incubator for 15–30 min to avoid condensation. The effects of very toxic compounds, such as staurosporine, can be seen immediately, so it is imperative to begin imaging less than 30 min–1 h after addition to cells. 12. The first time the apoptosis experiment is performed, a new image collection and processing definition need to be created.
Apoptosis and Cancer: Methods and Protocols by Gil Mor, Ayesha B. Alvero