Large-scale isolation of polymorphonuclear leucocytes from bovine blood

A method for the isolation of an enriched population (greater than 96%) of bovine polymorphonuclear leucocytes (PMNs) from 70 liters of blood was developed using a two-step procedure involving separation of the blood, in a packed red blood cell fraction containing the PMNs and a plasma fraction, by continuous flow blood separation. Hypotonic lysis of erythrocytes was then followed by centrifugation at 200 g sedimenting the PMNs. The yield was 93 +/- 30 g, the recovery was 62 +/- 20%, viability was greater than 95%. Since bovine blood can be obtained in unlimited amounts, the procedure described here can be applied to obtain large amounts of bovine PMNs for incubation studies and large-scale purification of intracellular enzymes suitable for biochemical characterization.     

A rapid method for the preparation of the neutrophil fraction of granulocytes from human blood by centrifugation on isotonic Nycodenz gradients

A rapid method for the preparation of neutrophils of the granulocyte fraction of human blood is described. A leucocyte-rich fraction is loaded onto preformed isotonic Nycodenz gradients, formed by mixing a stock isotonic Nycodenz solution with a NaCl diluent solution. After low speed centrifugation a neutrophil fraction of high viability, yield and purity can be isolated from the gradients.     

A rapid method for the isolation of coagulation factor XII from human plasma

Zinc chelate affinity chromatography was used to develop a rapid, three-step procedure to isolate coagulation factor XII from human plasma. The first step was ammonium sulphate fractionction which gave a 2-fold purification and 90% recovery in the 25–50% saturation fraction. The second step was zinc chelate affinity chromatography which gave a 240-fold purification and 67.5% recovery. The third step was zinc chelate affinity chromatography again, but with the application of a pH gradient. The overall recovery of zymogen factor XII was 21.7% and the total purification was 1992-fold. The purified factor XII had an apparent molecular weight of 77 600 as determined by SDS-polyacrylamide gel electrophoresis and a specific activity of 50 units/mg on a clotting assay.     

Biochemical characterisation of porcine polymorphonuclear leucocytes: comparison with human polymorphonuclear leucocytes

Polymorphonuclear leucocytes were isolated from pig blood relatively free from other cells and were characterised biochemically and morphologically and compared with human PMNLs. The activities of 16 enzymes of porcine and human PMNLs were measured and compared. Alkaline phosphatase, acid phosphatase, phosphodiesterase, gamma-glutamyl transpeptidase, NADH-cytochrome c oxidoreductase, malate dehydrogenase and acetylcholinesterase had higher specific activities in procine than in human cells. Alkaline phosphatase has an 87-fold higher specific activity in porcine than in human cells. beta-glucuronidase, lysozyme, beta-galactosidase, N-acetyl-glucosaminidase, beta-glucosidase, myeloperoxidase and catalase had higher specific activities in human than in porcine cells. beta-glucuronidase and myeloperoxidase showed over a 1000- and a 13-fold higher specific activity, respectively, in human than in porcine cells. Porcine PMNLs are readily available in large numbers and are recommended for studies of phagocytosis, chemotaxis and membrane biochemistry.     

A simple rapid method for the removal of leukocytes from human blood

Filtration of human blood cells through lamb''s wool columns removed more than 96% of all leukocytes in a series of experiments, while the retention of erythrocytes by the column averaged 6.4%. This method should prove extremely useful for obtaining pure erythrocyte preparations for use in biochemical and physiological studies, and for removing leukocytes from blood prior to transfusion.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure     

Leucocyte energy metabolism. VI. Simultaneous and improved isolation of lymphocytes and polymorphonuclear leucocytes from a single of human blood.

A technique, which combines a Ficoll-Rompacon treatment, sedimentation in a Dextran medium and erythrolysis by NH4C1, permitting the simultaneous isolation of polymorphonuclear cells (PMN) and lymphocytes from a single sample of human blood is described. The contamination of the PMN suspensions by other blood cells, including other categories of white cells, is minimal. However, the lymphocyte preparations, free of other white cells, are still contaminated by a non-negligible amount of thrombocytes. It could be shown that the biochemical system studied, the functional behavior and the morphological state of these cells are similar to that of white cells purified with the procedures previously used. Further, a numeration technique, based on DNA estimation, is presented which can be used when the cell count becomes inaccurate by cell agglutination.     

A rapid method for the isolation of human peripheral null lymphocytes.

We describe here a new technique for the isolation of human peripheral null lymphocytes: cells lacking detectable surface immunoglobulin as well as receptors for sheep erythrocytes. The double rosette technique described employs a combined negative selection for Ig⁺ cells by specific anti-Ig-coated sheep erythrocytes and E rosette selection for T cells. This singlestep procedure facilitates the isolation of null cells from large cell populations and can be completed in less than 2 hr. The isolated null subpopulation represents less than 5% of the total peripheral mononuclear cells and is at least 85–90% pure by sensitive surface marker analysis. The null subpopulation is shown to be highly enriched for effectors of both antibody-dependent cytotoxicity against autologous lymphocytes and spontaneous cytotoxicity against the K562 leukemia blast cell line. Application of this technique will allow further characterization of the effector populations for ADCC and NK as well as differentiation of T and B cell precursors.     

A method for the rapid isolation of virus from cultured cells

A simple and efficient collection method using hypotonic burst to isolate virions from infected cultured cells is described. Distilled water treatment of avian metapneumovirus (AMPV)-infected cells with thorough mixing and repeated pipeting was considerably faster for virion collection in avian cells compared to the widely used freeze-thaw (F-T) method (30 min vs. 3-4 h). This method was also more effective for virion collection. The total number of virions recovered from AMPV-infected immortal turkey turbinate cells by the novel water lysis method was 3-fold higher than by the F-T method. This simple water lysis method can be applied to virion collection for other RNA viruses such as the paramyxoviruses that are used to infect cultured cells.     

A rapid method for the isolation of peroxisomes from rat liver

A preparative method for the isolation of peroxisomes from the liver of normal, untreated rats is described. The peroxisome-enriched "light mitochondrial" fraction is layered on a 30% Nycodenz (5-[N-2,3-dihydroxypropylacetamido]-2,4,6-triiodo-N,N'-bis[2, 3-dihydroxypropyl]isophthalamide) solution containing 1 mM tetrasodium EDTA and then centrifuged in an angular rotor for 1 h at 130,000gavg. Peroxisomes are sedimented to the bottom leaving other organelles at the top of the tube. On the basis of morphological and biochemical studies, it is found that the peroxisomes (marker-enzymes catalase and urate oxidase) obtained in this method are not contaminated with lysosomes (marker-enzyme acid phosphatase) and contained very few mitochondria (marker-enzyme succinate-cytochrome c reductase) and microsomal vesicles (marker-enzyme glucose-6-phosphatase).     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

A simple method for human peripheral blood monocyte isolation

We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.     

A rapid method for the isolation of purified amyloplasts from wheat endosperm

A rapid method for the isolation and purification of amyloplasts from the endosperm of developing grains of Triticum aestivum L. has been developed. Cell-free amyloplasts were mechanically isolated from plasmolysed tissue, and then purified by low-speed centrifugation through a single layer of Nycodenz sedimenting onto a cushion of agar. Recovery of amyloplasts was greater than 20% with less than 1% contamination by cytosol, 0.2% by mitochondria, 0.5% by endomembrane system and no contamination by microbodies. This method yields preparations which are routinely 55–65% intact up to 2 h after extraction. Amyloplast integrity was shown to depend upon the external sorbitol concentration, and amyloplastic enzymes in intact preparations were protected from digestion by trypsin.Abbreviations APPase alkaline pyrophosphatase - BSA bovine serum albumin - Hepes 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid - (PPi)PFK pyrophosphate; fructose-6-phosphate 1-phosphotransferase Financial support for this research was provided by the Science and Engineering Research Council. The authors gratefully acknowledge many helpful discussions and initial assistance for this work from Professor T. ap Rees, Botany School, University of Cambridge, UK.     

A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

The metal ion requirement for activation of latent collagenase from human polymorphonuclear leucocytes

Latent human PMN leucocyte collagenase (enzyme-inhibitor complex) was shown to require zinc for the property of being activatable by various disulfides [see Macartney, H.W. and Tschesche, H. (1980) FEBS Lett. 119, 327--332]. The active enzyme also requires zinc for activity, indicating a possible participation in the enzyme's reaction mechanism and/or stabilization of the active site. The zinc in the latent enzyme may be removed by dialysis against EDTA, or cysteine. This produces a zinc-free latent enzyme which cannot be activated by any of the disulfide-containing activators. Readdition of zinc to the EDTA-inhibited latent enzyme, at the same concentration as the EDTA, produces an activatable latent enzyme once again. However, excessive zinc concentrations (more than three times the concentration of EDTA) exhibited an inhibitory effect on the activation process. Thereafter the inhibitor cannot be removed by disulfides from the enzyme-inhibitor complex of the latent enzyme. The zinc in the latent enzyme may be replaced by other double-positive metal ions such as cobalt, manganese, magnesium and copper.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.