By Perpetua M. Muganda
This unique quantity presents a unmarried, worthy reference resource for tactics that definitively determine and effectively quantify apoptosis. The publication starts with universal equipment applied to notice and quantitate apoptosis, in addition to apoptosis signaling pathways in toxicological and different comparable learn. It maintains with multi-parametric and phased apoptosis assays for detecting early and overdue apoptosis or distinguishing apoptosis from necrosis and autophagy. next chapters concentrate on fresh advances in genuine time and high-throughput assays that realize and quantitate apoptosis and apoptosis signaling pathways. ultimate chapters specialize in fresh advancements in preclinical anticancer therapeutics focusing on apoptosis. Written for the Methods in Pharmacology and Toxicology sequence, chapters function step by step descriptions of the methodologies, in addition to specialist assistance and implementation advice.
Vital and authoritative, Apoptosis equipment in Toxicology serves beginner scientists in addition to specialists, using a number of tools from universal laboratory gear to high-end dear and automatic equipment able to appearing genuine time apoptotic measurements.
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Additional info for Apoptosis Methods in Toxicology
The protocol described below is for control and DEB exposed lymphoblasts that grow as suspension cells. The notes [5–8] provide explanations, tips, and modifications for other cell Concurrent Phased Apoptosis Assays 39 Apoptosis Levels % pSIVA Positive Cells 8 10 mM DEB Exposure (h) 6 4 2 0 - + 0 - + 3 - + 8 - + 12 - + 14 - + 16 - + 24 Fig. 1 Detection of apoptosis in DEB-exposed TK6 cells using the pSIVA-IANBD assay: Control and 10 μM DEBexposed TK6 cells were stained with pSIVA at 3 h, 8 h, 12 h, 14 h, 24 h, and 36 h post DEB-exposure, as described in the protocol.
Immunol Lett 61:157–163 Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469–477 Bicknell GR, Snowden RT, Cohen GM (1994) Formation of high molecular mass DNA fragments is a marker of apoptosis in the human leukaemic cell line, U937. J Cell Sci 107(Pt 9):2483–2489 Schwartzman RA, Cidlowski JA (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr Rev 14:133–151 Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.
2. Remove from the freezer and equilibrate the Caspase-3/7-Glo® substrate and buffer to room temperature; place in a room temperature shallow water bath for approximately 30 min. Be sure to wipe the container with laboratory wipes dampened with 70 % ethanol before opening the container. 3. Meanwhile (during the ~30 min substrate is equilibrating to room temperature), deliver 1 × 104 cells in 100 μl media to designated wells within the 96-well plate for each control and toxicant-exposed sample. This should be done as efficiently as possible in order to minimize the amount of time cells are at room temperature, thus avoiding interfering with the ongoing apoptosis process.