subsequent Protocols

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

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Immunology: B Cell Protocols

Describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. - [Read Assays for B Lymphocyte Function Protocols]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere.  Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Protocol describes the directed coupling of antibody to protein G-agarose via its Fc domain, and subsequent covalent cross-linking of the complex using dimethyl pimelimidate (DMP). - [Read Directed Orientation of Antibodies during Preparation of Antibody Affinity Resin Protocol]

Category: Cell Biology and Cell Culture

Flow assays offer visualization of cell adhesion under wall shear stress. Visualization of the different events of cell adhesion can be quantified by selective image acquisition and subsequent image processing. Flow assays are suited for adhesive events which occur very rapidly in a time scale shorter than that of most static adhesion assays. Also, events subsequent to the initial events can be studied such as cell stabilization and spreading giving some insight into the kinetics of cell-cell. - [Read Dynamic Flow Assay for Cell Adhesion in a Parallel Plate Flow Chamber]

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

Flow assays allow visualization of cell adhesion under well-defined wall shear stress. Visualization of the events of cell adhesion are quantified by selective image acquisition and image processing. Events subsequent to the initial events can be studied such as cell stabilization and spreading. John T. Patton~GlycoTech Corporation, Rockville, Maryland - [Read Dynamic Flow Assay in a Parallel Plate Flow Chamber]

Category: Histology Protocols: Fixation of Cells Tissues Protocol

Fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Great detailed guide with protocols. Anthony S-Y Leong. - [Read Fixation and Fixatives - A great guide]

Category: Avian Bird Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA). The dsRNA is injected into the spinal cord of the embryo. Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA).  The dsRNA is injected into the spinal cord of the embryo.  Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules.  It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

LCM utilizes an infrared laser integrated into a standard microscope. A transparent cap is attached to a thermoplastic transparent membrane which lies directly on the surface of a routinely prepared tissue section on a glass slide. The investigator examines the tissue section microscopically and activates the laser when the desired cells underlie the target. This in turn activates the membrane with subsequent binding and procurement of the cells of interest. - [Read Laser Capture Microdissection (LCM)]

Category: Histology Protocols: Laser Capture and Microdissection

Laser Capture Microdissection of Living in vitro Cells. This PDF describes a precise, rapid and convenient Laser Capture Microdissection (LCM) method for the positive selection of living adherent cells and the successful subsequent re-cultivation of homogenous sub-populations. Arcturus. - [Read Laser Capture Microdissection of Living in vitro Cells PDF]

Category: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Protocol describes the purification, quantification, and subsequent sequencing of amplified DNA fragments using PCR. Excess nucleotides are removed from the initial PCR products using spun columns, and the products are quantified using fluorometry. - [Read Nonradioactive Cycle Sequencing of PCR-Amplified DNA Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. This protocol describes the use of differential centrifugation to fractionate a mammalian liver homogenate but similar methods should be applicable to all mammalian tissues and cultured cells. - [Read Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions.  This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength.  After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Protocol is the second in a set of three, describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  For the purposes of FDD gene expression analysis, as well as any other RNA-based gene expression technologies, contaminating genomic DNA must be removed before reverse transcription and subsequent PCR. - [Read Removal of Genomic DNA from Total RNA for Use in Fluorescent mRNA Differential Display Protocol]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol II]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

Protocol for slide preparation for laser capture microdissection (LCM) for subsequent DNA, RNA, and protein analysis. - [Read Slide Preparation for Laser Capture Microdissection (LCM) Protocol]

Category: Primary Cell Isolation and Culture Protocols

Slide Preparation for Manual Microdissection for Subsequent DNA, RNA, and Protein Analysis. Manual microdissection and subsequent molecular analysis can be carried out on slides stained using standard hematoxylin and eosin methods. However, if cell types that are (or are not) expressing a specific protein are required for a study, then more advanced slide preparation methods such as Immuno-LCM may be utilized. - [Read Slide Preparation for Manual Microdissection Protocol]

Category: PCR Protocols: Standard PCR Protocol

Protocol describes how to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase.  It is the foundation for all subsequent variations of the polymerase chain reaction. - [Read The Basic Polymerase Chain Reaction Protocol]

Category: Primary Cell Isolation and Culture Protocols

This method enables the isolation of different populations of liver cells and describes their subsequent culture. - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]

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

This method enables the isolation of different populations of liver cells and describes their subsequent culture. - [Read The Isolation and Culture of Rat Hepatic Cells Protocol]