Download PDF by Myrtle A. Davis: Apoptosis Methods in Pharmacology and Toxicology: Approaches

By Myrtle A. Davis

ISBN-10: 0896038904

ISBN-13: 9780896038905

ISBN-10: 1592592791

ISBN-13: 9781592592791

Dr. Myrtle A. Davis has assembled a panel of state of the art scientists to explain their most sensible equipment for detecting, illuminating, and quantifying apoptotic mechanisms in a manner that's valuable for the layout of toxicology and pharmacology experiences. those state of the art concepts contain circulate cytometric, fluorometric, and laser scanning equipment for quantifying and characterizing apoptosis, in addition to protocols for using DNA microarray know-how, excessive throughput monitors, and ELISA. Immunocytochemical tools for measuring biochemical and molecular endpoints in tissue sections could be hugely necessary for these conducting experiences in complete animal versions rather than phone tradition platforms.

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Additional resources for Apoptosis Methods in Pharmacology and Toxicology: Approaches to Measurement and Quantification (Methods in Pharmacology and Toxicology)

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3. Confocal laser scanning microscope images obtained on Hepa-1 cells exposed to tumor necrosis factor-alpha (TNF-_) and actinomycin D to induce apoptosis. Hepa-1 mouse hepatoma cells were treated with TNF-_/ActD, 12 h later stained with CMX Rosamine and Mitotracker Green, and then analyzed by CLSM. (A) shows light collected with a 605 nm long pass (red) filter (showing CMX Rosamine fluorescence), (B) shows light collected with a 530/30 nm band pass (green) filter (showing Mitotracker Green fluorescence), and (C) shows light collected with a 460/40 nm band pass (blue) filter (showing NAD(P)H autofluorescence).

30 Poot et al. 9. Since the functional state of the mitochondria is to be monitored, it is advisable to keep cell suspensions at their optimal temperature (37°C) and to allow them to recover for a brief moment after harvesting. 10. Dye solutions decompose rapidly if exposed to light. 11. Dye concentrations in the range of 100–200 nM are recommended, because at higher concentrations nonmitochondrial staining may occur. 12. Cells stained with MitoTracker Green FM show maximal emission at 516 nm; cells stained with CMXRosamine show maximal absorption at 594 nm and emit maximally at 608 nm; they also exhibit significant absorption in the UV region of the spectrum and may be excitable with a mercury arc lamp.

To collect fluorescence from MitoTracker Green FM, use a bandpass filter centered around 530 nm; for CMXRos use a longpass filter above 630 nm (see Notes 5–8). 4. PROTOCOL 2: COMBINED ASSAY FOR REDUCED THIOL AND NAD(P)H LEVELS AND MITOCHONDRIAL MEMBRANE POTENTIAL This protocol takes advantage of the fact that NAD(P)H emits blue fluorescence upon excitation with UV light. NAD(P)H fluorescence can thus be simultaneously measured with the green and red fluorescence of MitoTracker Green FM and CMXRosamine, respectively.

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Apoptosis Methods in Pharmacology and Toxicology: Approaches to Measurement and Quantification (Methods in Pharmacology and Toxicology) by Myrtle A. Davis

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