By Sharad Kumar, Loretta Dorstyn (auth.), Peter Erhardt, Ambrus Toth (eds.)
The skill to discover and quantify apoptosis, to appreciate its biochemistry, and to spot its regulatory genes and proteins is essential to biomedical learn. In Apoptosis: tools and Protocols, moment Edition, professional researchers describe the recommendations to top examine the severe steps excited by the apoptotic procedure. awarded from a number of diversified examine views, the amount includes sections masking detection of apoptosis, detection of non-apoptotic cellphone demise, adjustments of apoptotic proteins in the course of apoptosis, the research of its significant regulators, in addition to research of apoptosis in several organs and in version organisms. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible protocols, and notes on troubleshooting and heading off identified pitfalls.
Comprehensive and state-of-the-art, Apoptosis: tools and Protocols, moment Edition constitutes a key technical connection with the numerous methodologies utilized in the sector, and provides newcomers and skilled researchers strong instruments to light up the phenomena of programmed phone death.
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Extra resources for Apoptosis: Methods and Protocols, Second Edition
Sample text
1 mg/mL, it is estimated that 5–50 fg of each protein is delivered to each cell. 17. If time-lapse experiments are required after microinjection of C8-Bid, then it is recommended that the cells should be immediately placed on the microscope stage and brought to the experimental temperature due to the relatively short time to cytochrome c release. 18. At 1 h after completion of the peptide transfection protocol approximately 90–100% of cells will have released cytochrome c if 10 mg of Bid or Bim BH3 peptide is loaded.
This problem is exacerbated when the gene of interest is strongly proapoptotic; in such cases, cells that lose the gene of interest but maintain drug resistance have an obvious growth advantage. IRES or 2A peptide strategies (9), in which both the gene of interest and the drug selection marker are expressed off the same mRNA, may aid generation of a stable cell line. 8. , cytosolic rather than mitochondrial), then it may be necessary to use limiting dilution to select clonal cell lines displaying the correct localization.
10. Many apoptotic stimuli induce necrosis at higher doses. Inhibiting caspase-dependent cell death using a caspase inhibitor such as qVD-OPH confirms that a given dose is inducing apoptosis. 11. Cells must be plated on glass to enable the cells to be visualized by confocal or fluorescence microscopy postinjection and to provide an even, flat surface, decreasing the chance of breaking the microinjection needle. Cells tend to adhere less well to glass surfaces than to plastic so we recommend coating the glass with fibronectin prior to plating the cells.