By Demetrius Matassov, Terri Kagan, Julie Leblanc (auth.), Hugh J. M. Brady (eds.)
A set of state-of-the-art ideas for detecting and quantifying apoptosis, knowing its biochemistry, and for picking the genes and proteins that keep watch over and hold it out. defined in step by step element, those quite simply reproducible equipment variety from stream cytometry and immunohistochemical tactics to kinase job assays, yeast two-hybrid screening, and the cloning of novel genes through differential expression. The protocols stick with the winning tools in Molecular Biology™ sequence structure, each delivering step by step laboratory directions, an creation outlining the primary in the back of the strategy, lists of apparatus and reagents, and pointers on troubleshooting and keeping off identified pitfalls. Apoptosis tools and Protocols constitutes a key technical connection with the numerous methodologies utilized in the sphere, in addition to supplying amateur and skilled researchers alike strong instruments to light up the phenomenon of programmed cellphone loss of life.
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Extra info for Apoptosis Methods and Protocols
4. take advantage of the fact that chromatin composed of fragmented DNA can be physically separated from intact chromatin. , detects double-stranded DNA cleavage at the single-cell level by using terminal deoxynucleotidal transferase (TdT) to add fluorescein isothiocyanate (FITC)-labeled dUTP at the cleavage site; positive cells are detected by microscopy or by flow cytometry. It is important to understand that a cell that has undergone apoptosis will eventually lyse. , DNA fragmentation is detected prior to chromium release and chromatin condensation is observed microscopically before nonvital dyes are taken up (9,10).
For multicolor analysis to detect target or effector cells separately, perform immunofluorescence cell surface staining prior to the TUNEL procedure and resuspending cells in PBS. It is important to use PE- or Texas red-, but not allopyocyanin-, conjugated antibodies for cell surface staining as these fluorochromes are not destroyed by the fixation steps used in the TUNEL procedure. 18. A commercial kit can be used in place of this protocol and following the manufacturer’s instructions; however, purchasing the individual reagents is much more economical.
1999) DRONC, an ecdysone-inducible Drosophila caspase. Proc. Natl. Acad. Sci. USA 96, 4307–4312. 16. 16 Hawkins, C. , Yoo, S. , Peterson, E. , Wang, S. , Vernooy, S. , and Hay, B. A. (2000) The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM. J. Biol. Chem. 275, 27,084–27,093. 17. , Fritsch, E. , and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 18.