By Demetrius Matassov, Terri Kagan, Julie Leblanc (auth.), Hugh J. M. Brady (eds.)
ISBN-10: 0896038734
ISBN-13: 9780896038738
ISBN-10: 1592598129
ISBN-13: 9781592598120
A set of state-of-the-art ideas for detecting and quantifying apoptosis, figuring out its biochemistry, and for choosing the genes and proteins that control and hold it out. defined in step by step aspect, those simply reproducible equipment diversity from stream cytometry and immunohistochemical techniques to kinase task assays, yeast two-hybrid screening, and the cloning of novel genes by way of differential expression. The protocols persist with the profitable equipment in Molecular Biology™ sequence structure, each supplying step by step laboratory directions, an creation outlining the main in the back of the method, lists of kit and reagents, and tips about troubleshooting and warding off recognized pitfalls. Apoptosis tools and Protocols constitutes a key technical connection with the numerous methodologies utilized in the sector, in addition to supplying amateur and skilled researchers alike robust instruments to light up the phenomenon of programmed phone dying.
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A suite of state of the art options for detecting and quantifying apoptosis, figuring out its biochemistry, and for settling on the genes and proteins that control and hold it out. defined in step by step aspect, those easily reproducible tools variety from movement cytometry and immunohistochemical strategies to kinase task assays, yeast two-hybrid screening, and the cloning of novel genes through differential expression.
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J. Exp. Med. 181, 1661–1672. 16. , Reutelingsperger, C. , Kuijten, G. , Keehnen, R. , Pals, S. , and van Oers, M. H. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415–1420. 17. , and Reutelingsperger, C. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51. Analysis of Chromatin Changes 43 4 Methods of Analyzing Chromatin Changes Accompanying Apoptosis of Target Cells in Killer Cell Assays Richard C.
9. Calculate percent specific fragmented DNA or lysis using the following formula: % specific fragmented DNA or lysis = expt – spont × 100 100 – spont where expt is the experimental value (with NK cells) and spont is the spontaneous value (with TCM alone) for percent fragmented DNA or lysis as calculated in steps 7 and 8. 10. By utilizing cells that are labeled with 125I-UdR or [3H]TdR, but not with 51Cr, it is possible to use this assay to quantify DNA fragmentation alone. 11. Spontaneous DNA fragmentation should not exceed 30% (see Note 7).
11. Spontaneous DNA fragmentation should not exceed 30% (see Note 7). 3. Quantification of DNA Fragmentation Using the [3H]TdR Release Assay or JAM Test In this protocol, as first described by Matzinger (15), target cells labeled in their DNA with [3H]TdR are harvested after the coincubation period with NK cells onto a fiberglass filtermat using a semiautomated cell harvester. DNA that has been fragmented passes through the filtermat whereas intact, chromosome-length, DNA does not. The level of target cell DNA fragmentation is therefore inversely proportional to the amount of [3H]TdR retained on the filtermat.
Apoptosis Methods and Protocols by Demetrius Matassov, Terri Kagan, Julie Leblanc (auth.), Hugh J. M. Brady (eds.)
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