Isolation And Culturing Of Human Hepatocytes

The liver is estimated to be approximately 2.6 of the human body weight, consisting of both parenchymal and nonparenchymal cells. The parenchymal cells, commonly known as hepatocytes, comprise the majority of cells of the liver (ap-prox. 60 by cell number and 80 by weight). These cells are the major sites of xenobiotic metabolism and often are the cells injured by chemical toxicants. The nonparenchymal cells include the endothelial cells, which line the sinusoidal space kupffer cells, which are...

Blurring the Lines Between Primary Secondary and Tertiary Screening

Drug screening has traditionally been divided into three phases primary, secondary, and tertiary. As a result of the dramatic increase in targets and compounds to be screened, traditional screening is currently undergoing a re-evaluation. In addition to the move to miniaturization and increases in throughput, increased efficiency is also called for. One way to increase efficiency is to obtain more information at an earlier stage of drug screening. Primary screening is largely the establishment...

B FCS Detection Technologies

Fluorescence correlation spectroscopy (FCS) takes advantage of differences in the translational diffusion of large versus small molecules 7,8 . Each molecule that diffuses through the illuminated confocal focus gives rise to bursts of fluorescent light quanta during the entire course of its journey, and each individual burst is registered. The length of each photon burst corresponds to the time the molecule spends in the confocal focus. The photons emitted in each burst are recorded in a...

Info

Figure 2 Dose-response of purified IL-8 RNA. Error bars represent the standard deviation of three experiments. the samples in an incubator to automate the assay for increased throughput of the screen. IV. IN SITU mRNA DETECTION BY ANTI-RNA DNA ANTIBODIES This ISH technique was applied for direct detection of mRNA expression in per-meabilized cells. The assay was validated by measuring the expression of the IL-8 gene in uninduced and IL-1-induced endothelial cells. A schematic of the assay...

Marcel

Essentially three types of labeling of macromolecules (direct, indirect, and semi-direct) can be done for HTRF Lance assays similar to labeling for FP 27 . In direct labeling, the donor lanthanide chelate and the acceptor allophycocyanin, rhodamine, or fluorescein are directly labeled on molecule(s) involved in the reaction. In indirect labeling, these donor and acceptor fluorophores are labeled to macromolecules involved in the secondary interactions such as antibody-secondary antibody binding...

Fluorescence Resonance Energy Transfer FRET

One way to increase sensitivity, or perhaps more appropriately, selectivity, is to utilize fluorescence resonance energy transfer (FRET) technology. This technique has seen increasing use in cell imaging, flow cytometry, and confocal microscopy. FRET is the physical process by which energy is transferred from an excited chromophore (donor) to another chromophore (acceptor) by means of intermolec- Figure 4 Titration curves of a compound found to inhibit MMP3 were prepared in a 96-well plate (100...

Hts Using Alginate Macrodroplets

Sodium alginate is the water-soluble salt of alginic acid, a copolymer of 1-4 linked a-D-mannuronic and P-L-guluronic acids, which are extracted from various seaweeds. Sodium alginate solutions have the desirable property of gelling upon contact with divalent cations, such as Ca2+ 26,29 . Ca-alginate has been used as a convenient means of encapsulating cells and enzymes. The physical properties, such as gel strength and porosity, can be controlled by appropriate formulation of the starting...

Miniaturization Of Cellbased Assays Luminescence And Fluorescence

In vitro mammalian cell-based assays constitute an important part of any biomo-lecular screening effort. The main advantage in using whole-cell-based assays is that a cell model may be predictive of the desired physiological response. Data obtained from a cell-based assay not only relates to the target of interest but can on occasion predict such parameters as compound membrane permeability, general cell toxicity, and cell type specificity and selectivity. The disadvantage of using a cell-based...

Modern Integrated Robotic Systems

These systems remain largely based upon the architecture that was originally developed by Zymark. Most progress has been made in the development of appropriate modules or devices and user-friendly graphical (mostly Windows-based) interfaces to system management and scheduling software. While in the original Zymate architecture a proprietary bus interface (the controller) was used exclusively for module interfacing, most systems today are based upon standard industry buses (ranging from RS-232...

M A R C B L

Dimerization of growth factor, cytokine, or erythropoietin receptor is a prerequisite for all downstream effects. A high-throughput dimerization assay for erythropoietin receptor was described 106 . The extracellular domain of the erythropoietin receptor (rEpoR) was expressed in E. coli. A modified version of this protein with protein kinase A substrate site (33P-rEpoR) incorporated into rEpoR was also expressed in E. coli and phosphorylated in vitro using PKA and y-33P ATP. The dimerization...

Alpha Screen

The amplified luminescent proximity homogeneous assay screen Alpha Screen developed by BioSignal, a Packard Bioscience Company, is a nonradioactive proximity assay using two proprietary 200 nm diameter latex donor and acceptor beads 64 . The beads are coated with hydrogel to prevent nonspecific interactions and self-aggregation and to provide a functionalized surface for cova-lent attachment and to retain dyes. The donor beads contain a photosensitizer that absorbs light at 680 nm laser...

Fluorescence Polarization

Although the theoretical principles of fluorescence polarization FP were described at the beginning of the century 43 , the application of FP to drug discovery, and especially to HTS, became popular only recently 10,44-46 . FP measurements permit analysis of molecular orientation and motion of intrinsically fluorescent or fluorophore-labeled molecules in solution. Therefore FP methodol ogy can be applied to different assays measuring direct binding, such as antibody-antigen interaction,...

References

Synthesis and automated detection of fluorescently labeled primer extension products. BioTechniques 26 96-101, 1999. 2. TS Angeles, C Steffler, BA Barlett, RL Hudkins, RM Stephens, DR Kaplan, CA Dionne. Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity. Anal Biochem 236 49-55, 1996. 3. T Burke, R Bolger, W Checovich, R Lowery. Measurement of peptide binding affinities using fluorescence polarization. In Phage Display of Peptides and Proteins. A...

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Drug Discovery Diagram

Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat General factors, general transcription accessory proteins Antibody, anti-RNA DNA PK, proteinase K Oligos, DNA o-nts complementary to target RNA. Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat General factors, general transcription accessory proteins Antibody, anti-RNA DNA PK, proteinase K Oligos, DNA o-nts complementary to target RNA. Figure 9 The figure shows data for three...

Functional Expression Of Heterologous Potassium Channels In Yeast

The use of Saccharomyces cerevisiae cells as host for heterologous expression of potassium channels was made possible by advances in the understanding of the regulation of potassium ion homeostasis 5,6 . High-affinity potassium uptake in yeast is mediated by a potassium uptake transporter encoded by TRK1 7 . TRK2 encodes a cation transporter with moderate potassium affinity that contributes significantly to potassium uptake 8,9 . Potassium uptake is determined in part by the electrogenic...

CytostarT Technology

14c Labeled Compounds

Cytostar-T scintillating microplates from Amersham International plc are standard 96-well format, sterile, tissue culture treated microplates. The Cytostar-T plate is a polystyrene plate with a transparent base coated with scintillant. Upon addition of radioactive tracer to cells grown in the bases of the wells, the scintillant generates light when the tracer is bound to the cell membranes or taken up by the cell due to the close proximity of the radioactive isotope to the scintillant at the...