concentrations Protocols

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

The basis of this procedure is that two specific cell type preparations may be isolated, exposed separately to various compounds over a range of concentrations, and the cytotoxicity of these determined.  Parameters deemed indicative of a cytotoxic effect include a reduction in de novo protein synthesis and decreased glucose and fatty acid metabolism.  A cytotoxic effect may indicate that a chemical is likely to be nephrotoxic in vivo. - [Read Isolated Rat Glomeruli and Proximal Tubules]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

During incubation period and the course of disease, SARS virus replicates and releases from cells. The level of virus varies greatly from sample to sample. At early stage of incubation period or during the late convalescent phase, the concentration of virus is very low in all kinds of samples. Virus is also detected at extremely low concentrations in plasma during... - [Read Kit for Rapid Enrichment of SARS Virus]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time with cells that have been washed free of extracellular dye. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+], for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium in RAW 264.7 Cells]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the optimization of imidazole concentrations for immobilized metal-ion affinity chromatography. Most samples from which histidine-tagged proteins are to be purified also contain endogenous protein contaminants that bind to the immobilized metal-ion affinity chromatography (IMAC) adsorbent.  Usually, these proteins bind more weakly than the histidine-tagged protein. - [Read Optimization of Imidazole Concentrations for Immobilized Metal-Ion Affinity Chromatography Protocol]

Category: Nucleic Acid Electrophoresis Protocols

Protocol for the preparation of electrolyte gradient gels. Electrolyte gradients are formed when buffers of different concentrations are used in the upper (low electrolyte concentration) and lower (high electrolyte concentration) chambers of the electrophoresis device. - [Read Preparation of Electrolyte Gradient Gels Protocol]

Category: Fungi Protocols

Serum concentrations of voriconazole should be measured in patients receiving this drug to ensure that therapeutic levels are being achieved. The assay will give an indication of whether suitable blood levels have been achieved. - [Read Protocol: Bioassay for Determining Voriconazole Levels in Blood]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for pulsed-field gel electrophoresis via contour-clamped homogeneous electric field gels. In CHEF gels, the electric field is generated from multiple electrodes, arranged in a square of hexagonal contour around the horizontal gel and clamped to predetermined potentials.  Using a combination of low field strengths, low concentrations of aragose, long switching intervals, and extended periods of electrophoresis, DNAs up to 5000 kb can be resolved. - [Read Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols

Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Liver Toxicity Assays Protocols

Flow cytometry is used to monitor drug-induced changes in DNA and protein contents of hepatocytes cultured at physiological oxygen concentrations. - [Read Rat Hepatocyte Flow Cytometric Cytotoxicitiy Test]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Li]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

In an attempt to accurately measure DNA content with simultaneous preservation of cell surface markers, we have utilized gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association. For some cell types, the presence of apoptotic cells based on reduced DNA content can also be detected. One such technique employs the addition of ethanol to cells previously resuspended in high concentrations of fetal bovine serum... - [Read Simultaneous Analysis of DNA Content and Surface Immunophenotype Protocol]

Category: Mass Spectrometry Protocols

The dried-droplet method will fail for samples containing significant concentrations of involatile solvents or high concentrations of salts. PROWL - [Read Slow crystallization Protocol for MALDI samples]