By Larry A. Sklar
ISBN-10: 0195152344
ISBN-13: 9780195152340
Move cytometry is a delicate and quantitative platform for the size of particle fluorescence. In circulate cytometry, the debris in a pattern circulation in unmarried dossier via a concentrated laser beam at charges of hundreds of thousands to hundreds of thousands of debris in line with moment. in the course of the time each one particle is within the laser beam, at the order of ten microseconds, a number of fluorescent dyes linked to that particle are excited. The fluorescence emitted from every one particle is amassed via a microscope goal, spectrally filtered, and detected with photomultiplier tubes. move cytometry is uniquely in a position to the suitable and quantitative molecular research of genomic series info, interactions among purified biomolecules and mobile functionality. mixed with automatic pattern dealing with for elevated pattern throughput, those positive factors make move cytometry a flexible platform with functions at many phases of drug discovery. frequently, the debris studied are cells, particularly blood cells; movement cytometry is used commonly in immunology. This quantity indicates how circulation cytometry is built-in into sleek biotechnology, facing problems with throughput, content material, sensitivity, and excessive throughput informatics with purposes in genomics, proteomics and protein-protein interactions, drug discovery, vaccine improvement, plant and reproductive biology, pharmacology and toxicology, cell-cell interactions and protein engineering.
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Extra resources for Flow Cytometry for Biotechnology
Sample text
Changes in calcium distribution in cells treated with calcium ionophores). To stain constituents inside intact cells, a probe must be capable of crossing the cell membrane, either by diffusion or by some form of active transport or carriermediated transport. Most vital stains are small molecules that are relatively lipid soluble and are either positively charged or electrically neutral at physiologic pH. High lipid solubility favors partitioning of dyes from aqueous media into the lipid bilayer phase of the cell membrane and into membranous or lipid-containing intracellular structures.
These distributions can be analyzed with mathematical models, but it is also possible to combine sorting with CFSE labeling to isolate cells from different gen- 32 FLOW CYTOMETRY FOR BIOTECHNOLOGY erations (46), which cannot be done reliably when PKH26 is used as a tracking dye. CFSE labeling has been used extensively for studies of lymphocyte activation and differentiation (23, 40, 75). Probes of Membrane Potential, pH, and Calcium Changes in cytoplasmic and mitochondrial membrane potential, intracellular pH, and calcium ion concentration are associated with cell activation processes mediated by ligand-receptor interactions.
In principle, a flow cytometer should be able to measure fluorescence at any wavelength longer than that used for excitation. However, the light transmission of optics used in most instruments is typically poor at wavelengths below 400 nm, and the pho- FLUORESCENT PROBES 19 tomultiplier tube detectors supplied as standard equipment often do not respond well at wavelengths above 700 nm. It is usually possible to replace a standard-issue photomultiplier tube with one with extended red sensitivity; it may be harder to deal with poor light transmission below 400 nm.
Flow Cytometry for Biotechnology by Larry A. Sklar
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