By Demetrius Matassov, Terri Kagan, Julie Leblanc (auth.), Hugh J. M. Brady (eds.)
A suite of state-of-the-art options for detecting and quantifying apoptosis, knowing its biochemistry, and for picking out the genes and proteins that control and hold it out. defined in step by step aspect, those comfortably reproducible tools variety from move cytometry and immunohistochemical systems to kinase task assays, yeast two-hybrid screening, and the cloning of novel genes through differential expression. The protocols keep on with the winning equipment in Molecular Biology™ sequence layout, every one providing step by step laboratory directions, an advent outlining the primary at the back of the strategy, lists of kit and reagents, and pointers on troubleshooting and keeping off identified pitfalls. Apoptosis equipment and Protocols constitutes a key technical connection with the numerous methodologies utilized in the sector, in addition to providing beginner and skilled researchers alike strong instruments to light up the phenomenon of programmed phone demise.
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Additional resources for Apoptosis Methods and Protocols
Navia, M. , et al. (1994) Structure and mechanism of interleukin1` converting enzyme. Nature 370, 270–275. 8. 8 Walker, N. P. , Talanian, R. , Brady, K. , Dang, L. , Bump, N. , Ferenz, C. , et al. (1994) Crystal structure of the cysteine protease interleukin-1` converting enzyme: a (p20/p10)2 homodimer. Cell 78, 343–352. 9. , Nicholson, D. , Fazil, K. , et al. (1996) The three-dimensional structure of apopain/ CPP32, a key mediator of apoptosis. Nat. Struct. Biol. 3, 619–625. 10. 10 Mittl, P.
J. (1992) Comparative evaluation of several DNA binding dyes in the detection of apoptosisassociated chromatin degradation by flow cytometry. Cytometry 13, 137–143. 11. , and Stockinger, B. (1994) Mechanisms of tolerance induction in major histocompatibility complex class II-restricted T cells specific for a blood-borne self-antigen. J. Exp. Med. 180, 2089–2099. 12. , Hotz, M. , and Traganos, F. (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795–808. 13. 13 Johnson, L.
Both live and dead cells take up acridine orange and Hoechst 33342, whereas only dead cells take up ethidium bromide and propidium iodide. AO intercalates into DNA, making it appear bright green, and binds to RNA in the cytoplasm, staining it red. Hoe binds only to DNA, making it appear bright blue. EB and PI intercalate into DNA, making it appear orange, but bind only weakly to RNA, which may appear slightly red. Thus a viable cell stained with AO + EB will have bright green chromatin and red cytoplasm, whereas a dead cell will have bright orange chromatin (EB overwhelms AO) and its cytoplasm, if it has any RNA remaining, will appear dark red.