Multiple Centrifugation Research Articles

A Modified Technic for the Preparation of Metaphase Chromosomes From Mammalian Cells Grown in Monolayers

The details of a method for the preparation of well spread metaphase chromosomes from human cells grown on cover slips in Leighton tubes is presented. Cells were inoculated and grown to 25–50% confluency. Colcemid was added to collect cells in metaphase. The monolayers were washed free of medium and treated with hypotonic solutions in the Leighton tubes in which they were grown. The coverslips were fixed and air dried and stained immediately or stored in the Leighton tube for future histochemical or immunofluorescence tests.This modification of standard chromosome harvest technics eliminates trauma to the cells caused by the manipulations previously employed. It prevents the loss of metaphase cells and cell breakage which occurs as a result of multiple centrifugations and the preparation of cell spreads on glass slides or coverslips required by standard harvest procedures. It provides information regarding mitotic indices, chromosome number and structure with relative speed, ease and less cost.

Magnetic ferrofluids for preparation of magnetic polymers and their application in affinity chromatography.

THE use of magnetic polymers as supports for biological molecules has created much interest1–5. The inherent advantages of such preparations are, in particular, the ease of recovery of these polymers by applying a magnetic field. Thus, when used as supports for immobilised enzymes, their easy retrieval from liquors containing colloids or undissolved solids should be of great practical value1–3. Magnetic polymers have recently been tested as an alternative to conventional radioimmunoassay technique, obviating the need for vertical rotation and for the time-consuming, multiple centrifugations required with conventional solid phase procedures4–5. In the established techniques for producing magnetic polymer particles, the attachment of the biomolecules had to follow the preparation of the specific magnetic material; furthermore most of these preparations have been non-porous, exhibiting rather poor capacity. We describe here a novel and general procedure using magnetic fluids which allows ‘post-magnetisation’ of polymers already substituted with biological molecules. The properties of such preparations have been tested primarily as affinity chromatography gels. These preparations showed unaltered biospecificity when applied in general ligand-affinity chromatography studies. The simplified separation possible due to the magnetic properties of the gels eliminates the usual centrifugation and column chromatography steps. The procedure of ‘post-magnetisation’ seems to be suitable for different polymer particles, both unsubstituted gels and those carrying immobilised ligands as affinity adsorbents or enzymes.

Magnetic solid phase enzyme-immunoassay

A sandwich non-competitive enzyme-immunoassay procedure using antigen or antibody cavalently linked to magnetic polyacrylamide-agarose beads has been developed. A magnetic rack was used to separate the beads from the liquid phase and therefore time-consuming multiple centrifugation was avoided. Comparative studies using various conjugates of peroxidase, glucose oxidase and alkaline phosphate with antibodies were performed. In regard to the accuracy and reproducibility of the enzyme immunoassay, best results were obtained with alkaline phosphatase labelled antibodies. The procedure allowed the measurement of the following lowest quantities of proteins: 8 ng IgG, 40 ng IgA, 70 ng IgM, 1 i.u of IgE and 20 ng of specific antibodies against BSA.

Solid-phase, magnetic particle radioimmunoassay

A solid-phase radioimmunoassay system has been developed based on the use of antibodies covalently linked to polymer-coated iron oxide (Enzacryl R). An electro-magnet is employed both to mix the particles during incubation (by switching the field on and off) and to separate the antibody-bound and free fractions. This obviates the need for vertical rotation and for the time-consuming, multiple centrifugations required with conventional solid phase procedures. The system is universally applicable and methods have been established for the assay of thyroxine, human placental lactogen and digoxin. The thyroxine assay was employed as a model and it was shown that the results obtained for serum samples correlated closely with those using a routine liquid-phase radioimmunoassay. The applicability of employing a second antibody linked to the iron oxide particles was also studied.

Plasma membranes of liver cells of the chick embryo. I. Isolation procedures

The technique of isolation consists of a combination of homogenization, differential centrifugation, gradient centrifugation and the multiple poolings of isolated fractions. Large numbers of embryos are used to provide 5–10 g of accumulated liver tissue from the younger embryos (7, 11 days). Contamination by components of red blood cells or nuclear envelopes is avoided. The tissues are homogenized as dilute suspensions in either isotonic Krebs-Ringer solution or isotonic sucrose solutions supplemented with magnesium. Intact red blood cells are separated from the homogenate. Initial fractions are separated by multiple differential centrifugations. Fluffy coats and supernatants are pooled sequentially to increase the final yield of membranes. Intact nuclei can be separated by centrifugal rotation at 100 000 × g for 2 h over appropriate sucrose solutions. The separation of the plasma membrane fraction is carried out by density gradient centrifugation at 150 000 × g for 2 h. Fractions isolated in either isotonic sucrose or saline appear to be similar. The cytochrome oxidase and glucose-6-phosphatase activities are negligible. β-Glucuronidase activity is exceedingly low. Mg 2+-dependent ATPase activity is enhanced by the presence of Na 2+. The isolation of plasma membrane of embryonic cells allows for a more direct assessment of membrane development, cell-contact phenomena and membrane function.

The attachment of glycolytic enzymes to muscle ultrastructure

AbstractThe glycolytic enzymes, lactic dehydrogenase and aldolase, usually thought to be freely dissolved in the sarcoplasmic matrix, are in good part attached to the muscle ultrastructure. This attachment becomes manifest when the enzyme activities and specific activities of the press juices of whole skeletal muscles (rabbit) are compared with those of minced muscles, all obtained by ultracentrifugation of the tissues at 40,000 xpm for 16 to 20 hours. Mincing causes a great increase in the activities, associated with a rise in the volume and protein concentration of the press juices. We interpret these increases to be due to the solution in the matrix of enzymes previously attached to the ultrastructure.The same conclusion is reached by a different method, which we call “washing the ultrastructure.” It consists in multiple centrifugations of whole skeletal muscles, and removal of press juices, alternating with periods of imbibition of a buffer (0.1 M phosphate at pH 7.5) too dilute to dissolve out the fibrous proteins. During the imbibitions enzymes diffuse out into the buffer not imbibed, which becomes an extract. After four centrifugation‐imbibition sequences in as many days nearly all of the fluid matrix has been replaced by buffer. Enzyme activities fall steeply in press juices and extracts until nearly all freely dissolved enzymes have been washed away. Homogenates of the pressed muscles then show activities which are about half of those found in the homogenates of unpressed control muscles. We conclude that the enzymes found in the homogenates of the pressed muscles have previously been attached to the ultrastructure.Similar experiments with heart muscle indicate that nearly all of these enzymes are normally attached to the ultrastructure. Press juices contain only traces of activity, even after the heart has been minced. A fraction of the enzymes is slowly detached during the centrifugation‐imbibition sequences, appearing mainly in the extracts.

腟脂膏の細胞診について-特に多層式重層遠心分離法の応用 ―特に多層式重層遠心分離法の応用

腟脂膏の細胞診について-特に多層式重層遠心分離法の応用 ―特に多層式重層遠心分離法の応用

A study on the Separation of Cancer cells, especially about the cytological diagnosis in the internal medicine by way of the multiple layer centrifugation method

A study on the Separation of Cancer cells, especially about the cytological diagnosis in the internal medicine by way of the multiple layer centrifugation method

A Simple Method for Paraffin and Plastic Embedding of Protozoa

SUMMARY. To avoid multiple centrifugation and considerable losses of material in preparing protozoa for paraffin and plastic embedding a simple method has been worked out, requiring only two centrifugations, one before and one after fixation. During the second centrifugation, which is done at 700 g for 10 minutes, the organisms form a cohesive pellet which may be removed from the centrifuge tube by means of a fine spatula and handled as a piece of tissue through the whole process of dehydration and embedding.