Microscopic fluorescence assay

The selection of the type of assay plate is also dependent on the type of signal(s) to be detected and whether a clear bottom is required to observe cells under a microscope. Opaque black plates are often chosen for fluorescent assays and white plates are best for luminescent assays because of... 

Another morphological assay of apoptosis is done with acridine orange, a nuclear staining that reveals chromatin condensation under light and fluorescent microscope. 

Indirect immunofluorescence assay (IFA) A laboratory test used to detect antibodies in serum or other body fluid. The specific antibodies are labeled with a compound that will make them glow a fluorescent green color when observed microscopically under ultraviolet light. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

P 75] A static enzyme assay experiment was carried out using a stopped-flow method [161]. This is commonly used for monitoring reaction kinetics. P-Galacto-sidase was used as model enzyme to convert the substrate fluorescein mono-p-D-galactopyranoside (FMG) via hydrolysis into fluorescein. As buffer solution 10 mM potassium phosphate at pH 7.2 with 1 mM ascorbic acid was used to minimize photobleaching. The enzymatic reaction is accompanied by a change in fluorescence intensity which can be monitored with a microscope. 

This test, called the Comet Assay or single-cell gel-electrophoresis assay, allows the degree of DNA damage to be determined within a nucleic cell population. The principle of the method is based on the microelectrophoresis of nuclei of isolated cells, under basic conditions, on agarose gel (the whole being observed under a fluorescence microscope). 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

Function of mitochondria is also commonly monitored as an indicator of cellular toxicity. Mitochondrial uptake and retention of the fluorescent dye, rhodamine 123, can be visualized microscopically. Biochemical measurements of mitochondrial function include the ATP-ADP ratio and dehydrogenase activity with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which yields a colored formazan product upon reduction. The dye, neutral red (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), targets lysosomes, and its retention is inversely related to cytotoxicity. Commercially available versions of the MTT and neutral red assays have been adapted to microtiter plate formats to provide highly efficient screening assays. Examples of how cell-type-specific functions can be followed as indicators of cell toxicity are included in Table 8.1. 

HCS instruments take microscope-based images of a limited region of each microtitre plate well with sub-cellular resolution. Proteins of interest can be detected by a fluorescent tag, such as GFP, or are identified by fluorescent antibodies. One of the characteristics of HCS assays is the ability to not only monitor the levels of different proteins within the cells but also their location, the morphology and shape of the cells by multiplexing several labels.90 92... 

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