By Gil Mor, Ayesha B. Alvero
In Apoptosis and melanoma: equipment and Protocols, Second Edition, specialist researches within the box aspect the functionality of molecular and mobile biology suggestions for learning and detecting the activation of the apoptotic pathway. Chapters concentrate on assays constructed to discover its activation not just in vitro but additionally in vivo, optimized multiplex research, medium- to high-throughput displays, and the mobile approach. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and key tips about troubleshooting and keeping off recognized pitfalls.
Authoritative and useful, Apoptosis and melanoma: equipment 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 research and detection of apoptosis inside of and out of doors the world of melanoma learn.
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Additional resources for Apoptosis and Cancer: Methods and Protocols
ApopTag® kits detect single-stranded  and double-stranded breaks associated with apoptosis. Drug-induced DNA damage is not identified by the TUNEL assay unless it is coupled to the apoptotic response . The ApopTag® technique is helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing a DNA smear. However, it is important to evaluate TUNEL staining results in conjunction with morphological criteria. Although DNA fragments can be identified on agarose gels, the single-cell sensitivity of ApopTag® histochemistry is considered to be a far superior technology.
The proteasome inhibitor, bortezomib, is used in this model with the myelogenous leukemia cell line, K562. This protocol is written for cells cultured in suspension but can be amended to explore the kinetics of caspase activation using nearly any cell line, treatment, or test article. Attachmentdependent cells should be added to plates and allowed to recover/ equilibrate at least 2–16 h prior to dosing. Fig. 2 A real-time cytotoxicity induction time course. Bortezomib was twofold serially diluted in RPMI 1640 + 10 % FBS and added to replicate wells of a sterile opaque white plate in 50 μl.
Niles A, Worzela T, Scurria M, Daily W, Bernad L, Guthmiller P, McNamara B, Rashka K, Lange D, Riss T (2006) Multiplexed viability, cytotoxicity and apoptosis assays for cell-based screening. Cell Notes 16:12–15 9. Niles A, Moravec R, Riss T (2008) Update on in vitro cytotoxicity assays for drug development. Expert Opin Drug Discov 3:655–669 10. Leist M, Jaattela M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Biol 2:1–10 11. Worzella T et al (2013) Real-time cytotoxicity analysis using CellTox™ green cytotoxicity assay and the Tecan Infinite® 200 PRO with gas control module.