density Protocols

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Protocol describes systems in which the membranes are allowed to float through a density gradient from a dense load zone. This is an ideal format. - [Read A Self-generated Gradient to Discriminate a Cytosolic or Membrane Location of Large Protein Complex]

Category: Protein Protocols: Protein Trafficking

Protocol is based on methods for the resolution of GLUT4 containing vesicles and the identification of phosphoinositide kinase containing vesicles in 3T3-L1 adipocytes. They may have a wider application to any low-medium density membranes. Protocol incorporates the strategy of using a low density microsome fraction as the gradient input, commonly used in GLUT 4 studies that may have a wider application to other investigations. - [Read Analysis of Membrane Trafficking and Intracellular Signaling in Self-Generated Iodixanol Gradients]

Category: Cell Biology and Cell Culture

OptiPrep Application sheet C14 describes procedures for determining the density and sedimenting properties of any cell (of any size or density) using either a continuous or discontinuous gradient of iodixanol. This Application Sheet describes procedures aimed at isolating specifically a relatively low-density cell fraction from any tissue. - [Read C25 Isolation from spleen, thymus, pancreas, alveolar tissue and other tissues]

Category: Cell Biology and Cell Culture

The protocol presented in this Application Sheet uses an alternative strategy to sedimentation on to a density barrier, that is to adjust the density of whole blood to a value just greater than the cells of interest and allow them to float to the surface. - [Read C8 Isolation of bovine peripheral blood mononuclear cells by flotation.]

Category: Cell Biology and Cell Culture

Cell fractionation techniques are presented for the design of gradient systems for separating one or more cell types from lavages of body cavities or from mechanically or enzymically-dissociated tissues. Includes: Preparation of cell suspension for gradient loading; Fractionation by buoyant density; Fractionation on the basis of cell size. - [Read Cell Fractionation of a Mixed Population of Cells]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

The use of Ferbach flasks is an ideal method to amplify the cells and virus inoculum for the bioreactor. The method is also very practical for producing sufficient amounts of various proteins for feasibility studies using HyQ© SFX-Insect™ medium. The large surface area in this vessel enables sufficient aeration and, therefore, the cells, when they reach high density, are not oxygen depleted. - [Read Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter]

Category: Transfection Protocols

Protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. - [Read DNA Transfection Mediated by Lipofection Protocol]

Category: E Coli Protocols: E coli Nucleic Acid Protocols

E.coli total RNA labeling protocol for high density oligonucleotide array. Includes: RNA Preparation; Digest RNA and Purification of cDNA; Purify cDNA with Qiaquick PCR purification kit; cDNA Fragmentation and end labeling; Labeling with Terminal Transferase. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol for e.coli total RNA labeling for high density oligonucleotide array. - [Read E.coli Total RNA Labeling Protocol for High Density Oligonucleotide Array]

Category: Cell Biology and Cell Culture

Cell fractionation of cellular components using Percoll a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. Percoll becomes a simple matter to establish a linear density gradient. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients. - [Read Equilibrium Density Gradient Percoll Protocol]

Category: Animal Research Techniques

Needs of animals, Ventilation, lighting and temperature considerations. Cage size/density. Handling. Blood Collection and guidlines. EXCELLENT GUIDE GC470 ESSENTIALS FOR ANIMAL RESEARCH BRL Univ. Illinois. - [Read Ethics, Guidelines for Animal Research PDF]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Feeder Cell Density - A key Parameter in Human Embryonic Stem Cell Culture - [Read Feeder Cell Density - A key Parameter in Human Embryonic Stem Cell Culture]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

The protocol described in this protocol has been used principally for analyzing the Golgi, endoplasmic reticulum and trans-Golgi network but markers for other compartments (e.g. ERGIC and endosomes) have also been analyzed. Modifications either to the gradient density range or the centrifugation conditions influence the ability of the gradient to resolve multiple compartments. - [Read Fractionation of Golgi, ER, TGN and Other Membrane Compartments in Pre-Formed Iodixanol Gradients]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

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

Generation of a growth curve can be useful in evaluating the growth characteristics of a cell line. From a growth curve, the lag time, population doubling time, and saturation density can be determined. - [Read Generation of Growth Curve Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment. - [Read Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol]

Category: Cell Biology and Cell Culture

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations. Reduction of the amount of microbeads reduces the costs of the experiment. - [Read Isolation of monocytes from monocyte enriched PBMC fraction using CD14]

Category: Yeast Protocols

Simple method for determining the cell density of yeast cultures. - [Read Measuring Yeast Cell Density by Spectrophotometry 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 Protocols

Protocol is used to prepare the arms of any bacteriophage {lambda} vector. - [Read Purification of Bacteriophage lambda Arms: Centrifugation through Sucrose Density Gradients Protocol]

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

A solution containing plasmid DNA, saturating amounts of ethidium bromide, and CsCl (44% w/v) is layered between two solutions of lesser (35% w/v CsCl) and greater density (59% w/v CsCl).  During centrifugation to equilibrium, the closed circular plasmid DNA and linear DNAs form bands at different densities. - [Read Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient]