Search results for: 96 Well Microplates for Colorimetric Assays
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Comparison between Folin-Ciocalteu and Prussian Blue Assays to Estimate The Total Phenolic Content of Juices and Teas Using 96-Well Microplates.Folin-Ciocalteu colorimetric assay (FC) is the most widely used assay to estimate the total phenolic content in foods, beverages, herbs and other plant extracts, but many chemical compounds may act as interfering agents, producing inaccurate estimations of the real concentration of phenolic compounds in the matrix. Based on this limitation, the objective of this study was to compare, quantitatively, the Folin-Ciocalteu and Prussian Blue (PB) assays in estimating the total phenolic content in purple grape juices (n = 20; Vitis labrusca L.) and teas (n = 25) from different botanical origins using 96-well microplates. PB assay presented a low limit of detection (PB = 0.27 mg/L; FC = 0.25 mg/L) and quantification (PB = 0.92 mg/L; FC = 0.82 mg/L), showing its suitability in screening the total phenolic content in grape juices and teas. FC and PB assays presented a high association (P < 0.0001) for teas (r = 0.887) and grape juices (r = 0.923). The advantages of PB over FC assay are its simplicity, low time consumption (15 min reaction as compared to 60 min reaction for the FC assay), lower usage of reagents (solutions are prepared in a mM base), and higher selectivity. Additionally, PB assay was proven to be reproducible and repeatable and, therefore, may be used as an alternative to FC assay.
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Determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays.Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.
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Acetylcholinesterase inhibitory activity of uleine from Himatanthus lancifolius.Application of acetylcholinesterase (AChE) inhibitors is the primary treatment for Alzheimer's disease. Alkaloids, such as physostigmine, galanthamine, and huperzine A, play an important role as AChE inhibitors. The aim of this work was to evaluate Himatanthus lancifolius (Muell. Arg.) Woodson, a Brazilian species of Apocynaceae, and its main indole alkaloid uleine, in order to identify new AChE inhibitors. The plant fluid extract, fractions, and uleine were tested for AChE inhibitory activity using Ellman's colorimetric method for thin-layer chromatography (TLC), 96-well microplates, and also Marston's TLC colorimetric method. Both TLC assays showed similar results. At 5 mg/mL, the fluid extract inhibited the AChE enzyme by (50.71 +/- 8.2)%. The ethyl acetate fraction exhibited the highest level of AChE inhibition, followed by the dichloromethane fraction. The isolated alkaloid uleine displayed an IC50 value of 0.45 microM.
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Cytotoxic effects of a mixed ligand copper(II) chelate complex against a panel of human and murine cancer cells in vitro. Theoretical study of the mechanism of biologic action through molecular modelling.In an effort to discover new compounds with anticancer activity, we have developed a novel copper (II) [Cu(II)] chelate complex with a tridentate ONNSchiff base ligand and the anion of salicylate and we evaluated the in vitro chemosensitivity of various human and murine tumor cell lines by measuring cell growth inhibition. The ultimate goal was to evaluate the existence of a potential antitumor activity of this complex. Beyond the cytotoxic activity assessment of the complex, we aimed at the elucidation of the underlying mechanism of action of this complex and its interactions with biological molecules, carrying out theoretical (quantum-chemical) calculations.
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Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.
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Colorimetric and fluorimetric microplate assays for legumain and a staining reaction for detection of the enzyme after electrophoresis.The cysteine endopeptidase legumain was recently discovered in mammalian cells, predominantly localized in the lysosomal system where it is believed to contribute to antigen processing for MHC class II. Here we describe rapid assay procedures for the enzyme in 96-well microplates with two substrates, a novel compound, succinyl-Ala-Ala-Asn-4-methoxy-2-naphthylamide, and benzyloxycarbonyl-Ala-Ala-Asn-4-methyl-7-coumarylamide. Both substrates are suitable for fluorimetric assays, but the naphthylamide also allows colorimetric detection of legumain activity, since the released 4-methoxy-2-naphthylamine gives a red product when coupled with the Fast Garnet color reagent. We show that the naphthylamide substrate can be used to visualize active legumain after electrophoresis in polyacrylamide gel. Both substrates provide assays that are reproducible and sufficiently sensitive to allow the assay of legumain in crude samples such as tissue homogenates, although the coumarylamide is the more sensitive. The specificity of both assay methods for legumain was verified by the lack of inhibition by E-64 and total inhibition by egg white cystatin.
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[Cell culture and its application--in vitro evaluation of anticancer activity using human tumor cell lines].Selective toxicity against cancer cells is a most important determinant for anticancer agents. Therefore, we have preferably evaluated anticancer effects in vivo using murine tumor models for several decades. Approximately 50 anticancer agents are currently available for clinical therapy, but very few agents are effective against some types of cancer. Much progresses in cell culture techniques resulted in establishment of various human tumor cell lines. Currently, we are able to use human tumor lines as well as murine ones for the examination of drug sensitivity. A number of assay methods to evaluate anticancer activity have been developed. In the beginning, growth inhibitory activity was evaluated by counting cell numbers after drug exposure. Then, human tumor clonogenic assay (HTCA) was designed to measure only proliferative cells. Recently colorimetric MTT assay and SRB assay in 96-well microplates were developed, which were adopted in the screening system in the NCI, based on a new idea, that is, disease-oriented screening (DOS) using about 60 human tumor cell lines. In this paper outline of each method was described, adding especially several comments on disease-oriented screening.
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A colorimetric assay for the simultaneous measurement of plasminogen activators and plasminogen activator inhibitors in serum-free conditioned media from cultured cells.The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted for use with 96-well plates and an automatic microplates spectrophotometer. The assay allows the discrimination between tissue-type and urokinase-type plasminogen activators in cell culture-conditioned media. It provides a level of detection of these enzymes in the range 10(-17) to 10(-13) mol (determined using purified human plasminogen activators), uses no radioisotopes, and is faster and more economical than similar assays using specific peptide substrates for plasminogen activators. Levels of free plasminogen activator inhibitor activity can be simultaneously measured on the same samples by a simple adaptation of the assay. This method allows an easy treatment of the data by interfacing with a computer and should thus be useful when large numbers of samples are assayed.
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A rapid, automated colorimetric assay for measuring antibody binding to cell surface antigens.An automated, colorimetric procedure is described for detecting antibodies specific for cell surface antigens. The procedure entails (a) coating the wells of 96-well microplates with either protein A or anti-immunoglobulin antibodies and (b) preincubating either the microplate or target cells with the test antibody. Target cells which react with the test antibody bind to the wells of the microplate and bound cells are quantitated by staining with the dye Rose Bengal. A microplate spectrophotometer is used to measure absorbance in each well of the plate, providing a rapid, automated measure of antibody titre. The assay is simple to perform, uses readily available reagents and gives comparable sensitivity to rosetting assays. With these features, and the capacity for handling large numbers of trays quickly, this method has obvious advantages in screening for antibody activity in culture supernatants of hybridoma clones.
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Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.
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