vivo Protocols

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes typical methods that are used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read A Protocol for AAV Vector Production and Purification]

Category: Cloning Protocols: Vector Protocols

Protocol is used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read AAV Vector Production and Purification Protocol]

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

Category: Mouse Protocols

Protocol for calcium dependent by pentraxins to ligands immobilized on agarose, and effect of C-reactive protein on leptin action in mice in vivo. Includes: Testing for binding of CRP to immobilized leptin; Testing for binding of radiolabelled leptin by immobilized CRP, SAP and leptin receptor; Testing for effect of human CRP on human leptin action in mice. - [Read Calcium Dependent by Pentraxins to Ligands, and Effect of C-reactive Protein on Leptin Action]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. - [Read Chromatin Immunoprecipitation and Microarray-Based Analysis of Protein Location Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Chromatin immunoprecipitation is a method for determining whether a particular protein binds to a particular DNA sequence in vivo. - [Read Chromatin Immunoprecipitation in Yeast Protocol]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP assay protocol with 2 steps: in vivo formaldehyde cross-linking of whole cells and protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Breeden Lab - [Read Chromatin IP (CHIP assay)]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Cell-based assays are important tools for contemporary biology and drug discovery because of their predictive potential for in vivo applications.This assay gives ratiometric, inversely proportional values of viability and cytotoxicity (Figure 4.15) that are useful for normalizing data to cell number. Also, this reagent is compatible with additional fluorescent and luminescent chemistries. - [Read Determining Number of Live and Dead Cells in Cell Population: Cytotoxicity Assay Protocol]

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Protocol for electroporation and RNAi in the rodent retina in vivo and in vitro. - [Read Electroporation and RNAi in the Rodent Retina In Vivo and In Vitro Protocol]

Category: RNAi Technology Protocols

Protocol for electroporation and RNAi in the rodent retina in vivo and in vitro. - [Read Electroporation and RNAi in the Rodent Retina In Vivo and In Vitro Protocol]

Category: Gene Delivery Protocols: Biolistics Protocols

The technique has many advantages—plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells. - [Read Gene Delivery to Skin Using Biolistics Protocol]

Category: Signalling Signal Transduction Protocols

This protocol assays inhibition of in vivo binding of [3H]-cAMP to cAR1 by GTPγS. Dictyostelium discoideum respond to extracellular cAMP through the cAMP chemoattractant receptor (cAR1). Binding of cAMP to the G protein-coupled cAR1 is inhibited by the GTP analog GTPγS. Protocol includes information on: Solutions used, BioReagents and Chemicals and Protocol Hints. - [Read GTPγS-Induced Inhibition of cAMP Binding to the cAMP Receptor (cAR1) in Dictyostelium Discoideum]

Category: Cell Biology and Cell Culture: Hepatocyte Cell Culture

Primary culture of hepatocytes is an in vitro model widely used to ... protocols for cytochromes P450IIIA in vivo and in primary cultures of animal and ...Human Hepatocyte Culture Protocol - [Read Human Hepatocyte Culture Protocol]

Category: Gene Delivery Protocols

This highly efficient in vivo gene transduction technique for laboratory mice. Hepatocytes are most effectively transduced by tail vein injection of a large volume of DNA solution in a short time. Practice with the injection technique is necessary!!! - [Read Hydrodynamics-Based Gene Transduction Protocol]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify novel interacting partners of a target protein. In both cases, the cells, which may have been labeled with [35S]methionine, are harvested and lysed under conditions that preserve protein-protein interactions. The target protein is specifically immunoprecipitated from the cell extracts, and the immunoprecipitates are fractionated by SDS-PAGE. - [Read Identification of Associated Proteins by Coimmunoprecipitation Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol for the immunoprecipitation (IP) of Homer 1a, injection of virions and in situ hybridization in the spinal cord. Includes: Immunoprecipitation (IP) of Homer1a from spinal cord; Injection of virions in the parenchyma of the spinal dorsal horn in vivo; Generation of cRNA probes; Analysis of DIG-dUTP incorporation; Tissue hybridization. - [Read Immunoprecipitation of Homer 1a, Injection of Virions and In Situ Hybridization in the Spinal Cord]

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

The effect of a test compound, in the presence and absence of S-9 mix, on the differentiation and growth of rat limb bud and CNS cells in vitro indicates whether it is potentially a teratogen in vivo. - [Read In Vitro Micromass Teratogen Assay]

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

The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDBK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs.  A correlation between in vitro cytotoxicity, as measured in this system, and LD50 values in rats and mice has also been established. - [Read In Vitro Prediction of the Maximum Tolerated Dose Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

Protocol for the in vivo actin polymerization. - [Read In Vivo Actin Polymerization (cAMP) Protocol]

Category: Cloning Protocols: Vector Protocols

Protocol for in vivo cloning with mu. - [Read In Vivo Cloning with Mu Protocol]

Category: Immunology: Immunoassays

Protocol for in vivo cytokine capture assay (IVCCA). - [Read In Vivo Cytokine Capture Assay (IVCCA) Protocol]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Positron emission tomography (PET) is a established quantitative and noninvasive imaging modality. With the PET reporter gene (PRG)/PET reporter probe (PRP) system, based on a mutant form of herpes simplex virus 1 thymidine kinase (HSV1-sr39tk), the PET signal is directly proportional to the enzymatic activity of sr39TK9-14. In this protocol, we describe in detail a method for reporter gene labeling of islets and quantitative scanning using a reporter probe. - [Read In Vivo Functional Real-Time Imaging of Transplanted Islets Using Positron Emission Tomography (PET)]

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: Gene Delivery Protocols: DNA Nanoparticles Protocols

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. - [Read Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol]