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Biomedical subjects

John M Graham

Publications and source records attributed to John M Graham.

At least 55 records · Page 3Linked to original sources
PubMed2002

Purification of lipid rafts from cultured cells.

Lipid-rich lipid rafts are microdomains of the plasma membrane that are resistant to low concentrations of nonionic detergent. This forms the basis for their isolation. Either a microsomal fraction or a postnuclear supernatant are loaded beneath a discontinuous iodixanol gradient. If all the solutions contain 0.5-1.0% Triton X-100, the intact lipid rafts float to the top of the gradient while all of the other detergent-solubilized membranes remain at the bottom.

Cell Fractionation↗
PubMed2002

Purification of islets of Langerhans from porcine pancreas.

Flotation through a slightly hyperosmotic discontinuous gradient iodixanol achieves a much higher recovery of islets of an improved viability than the customary method using sedimentation on to a diatrizoate/polysaccharide barrier. Flotation techniques achieve an enhanced separation of the islets from any residual digestive enzymes and from acinar cells. The method has been extended to human pancreas.

Animals↗
PubMed2002

Separation of human monocytes from a leukocyte-rich plasma.

Human peripheral blood monocytes are isolated by flotation from a dense leukocyte-rich plasma (LRP) through two lower-density barriers prepared from OptiPrep. The separation from lymphocytes depends on the more rapid rate of flotation of the monocytes because of their slightly lower density and larger size. The method works optimally only with fresh (within 2 h of drawing) EDTA-anticoagulated blood.

Antigens, CD↗
PubMed2002

Isolation of peripheral blood mononuclear cells from macaques on a density barrier.

The standard techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) using commercial "lymphocyte isolation media" cannot be satisfactorily extended to experimental animals without manipulating either the density or the osmolality of the medium. PBMCs from Macaques can also be isolated from whole blood by sedimentation on to a density barrier containing approx. 10% iodixanol, polysucrose (Ficoll) with a density of approx 1.074 g/ml.

Animals↗
PubMed2002

Purification of parietal and chief cells from the gastric mucosa.

Acid-secreting parietal cells from the gastric mucosa are widely studied as a model in studies on ion transport. A discontinuous gradient of iodixanol has been found to be superior to earlier protocols using Nycodenz and this method, which removes a significant amount of contaminating cells and mucus is a very useful prelude to further purification by elutriation.

Animals↗
PubMed2002

Isolation of rat and human hippocampal neuron fractions in a discontinuous density gradient.

The plating efficiency of neurons in culture is highly dependent on the concentration of cells used to establish the monolayer. A discontinuous iodixanol gradient permits both the production of a viable concentrated suspension of neurons and purification from other brain tissue elements. The gradient that is described in this Protocol Article is applicable to brain tissue from rat and also from human biopsy specimens.

Animals↗
PubMed2002

Preparation of crude subcellular fractions by differential centrifugation.

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. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes.

Animals↗
PubMed2002

Purification of gastric mucosal ECL cells from a crude elutriation fraction.

Acid-secreting parietal cells from the gastric mucosa are widely studied as a model in studies on ion transport and the endocrine/paracrine ECL cells effectively control parietal cell function. Discontinuous gradients of iodixanol for the purification of ECL cells were subsequently simplified to the use of a density barrier. This technique is now commonly used following initial centrifugal elutriation.

Cell Separation↗
PubMed2002

Homogenization of mammalian cultured cells.

Satisfactory homogenization of cultured cells is a necessary prerequisite to any fractionation schedule. Protocols are given for homogenization in iso-osmotic (A) and hypo-osmotic (B) media that should be broadly applicable to any cell type and to any subsequent fractionation procedure. Alternative procedures are also summarized in the Notes section, but detailed operation of some of the automated devices is beyond the scope of this short Protocol Article.

Animals↗
PubMed2002

Homogenization of Mammalian tissues.

Satisfactory homogenization of a tissue is a necessary prerequisite to any fractionation schedule. A detailed protocol is given for rat liver because of the widespread use of this tissue. Although this technique should be broadly applicable to any soft tissue and to any subsequent fractionation procedure, there are certain tissues and applications that require either minor or extensive modification. Some of these points are addressed in the Notes section.

Animals↗
PubMed2002

Isolation of human platelets (thrombocytes).

Platelets from human blood can be isolated in high yield by centrifugation of whole blood over an iodixanol density barrier of 1.063 g/ml. The separation from all of the blood cells (which form a pellet) is based on the slower sedimentation velocity of the smaller platelets.

Blood Platelets↗
PubMed2002

Separation of membrane vesicles and cytosol from yeast, cultured cells, and bacteria in a small volume self-generated gradient in a fixed-angle rotor.

There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from yeast, cultured cells, or from bacteria. This Protocol Article describes the flotation of the vesicles through a self-generated gradient from a dense sample zone using the low-viscosity medium iodixanol. As the sample is exposed to the gmax the tendency of the proteins to sediment overcomes any diffusion in the opposite direction and are therefore completely separated from the vesicles.

Animals↗
PubMed2002

Rapid purification of nuclei from animal and plant tissues and cultured cells.

Nuclei are isolated by buoyant density banding in a discontinuous iodixanol gradient, under isoosmotic conditions. The low viscosity of the gradient allows the purification to be carried out at 10,000g in only 20 min. The method avoids possible damage to nucleoprotein complexes caused by hyperosmotic sucrose gradients. Although developed for mammalian liver the method can be applied (with or without minor modifications) to any tissue or cell type.

Animals↗
PubMed2002

Separation of membrane vesicles and cytosol from cultured cells and bacteria in a preformed discontinuous gradient.

There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from either cultured cells or from bacteria. Flotation of the vesicles through a low-density barrier from a dense sample zone using the low viscosity medium iodixanol allows complete separation of these compartments. As the sample is exposed to the gmax the tendency of the proteins to sediment overcomes any diffusion in the opposite direction.

Animals↗
PubMed2002

Purification of peroxisomes in a self-generated gradient.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a self-generated gradient. Self-generated gradients make sample handling very easy and are highly reproducible but need to be formed in either a vertical, near-vertical, or small volume high-performance fixed-angle rotor. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll there is no contamination from endoplasmic reticulum.

Animals↗
PubMed2002

Purification of peroxisomes in a preformed iodixanol gradient in a fixed-angle rotor.

In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a preformed isosmotic continuous gradient. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll there is no contamination from endoplasmic reticulum.

Animals↗
PubMed2002

Separation of monocytes from whole human blood.

Human peripheral blood monocytes are isolated by flotation from whole blood through a single low-density barrier prepared from OptiPrep at 4 degrees C. The separation from lymphocytes depends on the more rapid rate of flotation of the monocytes because of their slightly lower density and larger size. The method works optimally only with fresh (within 2 h of drawing) EDTA-anticoagulated blood. Preliminary evidence suggests that this technique may be applicable to blood from rats.

Blood Cells↗