David Rickwood

Nycodenz: A new nonionic iodinated gradient medium

Abstract The properties of Nycodenz, a new nonionic iodinated density gradient medium, are described. This compound shares a number of characteristics with its related compound metrizamide. However, the stabler, more inert nature of Nycodenz endows it with a number of favorable properties as a gradient medium.

Isolation of the major subcellular organelles from mouse liver using nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.

Formation of isotonic Nycodenz gradients for cell separations

Abstract Nycodenz is a new nonionic iodinated gradient medium which readily dissolves in water to give nontoxic, autoclavable solutions. This paper describes the use of diffusion techniques to prepare isotonic Nycodenz gradients with maximum densities up to 1.15 g/ml, which is sufficient to band most types of cells.

Evidence of surface antigen detachment during incubation of cells with immunomagnetic beads

We have studied the attachment of immunomagnetic beads to different cells, with particular interest in cells that did not, as expected, appear to bind antibody-coated beads. Through the use of immunofluorescence and laser scanning confocal microscopy it was possible to demonstrate that beads can detach significant amounts of antigen from the surface of cells. This results in the appearance of antigen-depleted yet viable cells. Moreover, the detached antigen is found to be bound to beads and is associated with fragments of cell membrane which can also carry other (non-bead binding) cell surface proteins. After reculturing, antigen-depleted cells can recover their normal levels of surface antigen. Our results demonstrate the existence of an immunobead-induced cell membrane detachment phenomenon that can lead to the removal of all of a specific surface antigen without killing the cells, as judged by both vital staining and reculturing. An important aspect of this phenomenon is that immunoidentification of immunobead-selected populations of cells will give erroneous results. This may thus be of significance for the immunobead-based cell depletion methods that are used in medicine.

Biotechniques: Transfection of cells by immunoporation

Cell transfection is now a central technique in molecular biology and an essential prerequisite for gene therapy. Here we describe how beads coated with antibodies and bound to specific cell-surface transmembrane proteins can create holes in cells when the beads are removed, allowing transfection of the cells with DNA or other macromolecules. This unique targeted transfection of cells by immunoporation is very efficient and results in minimal cell death.

Isolation and preliminary characterisation of DNA-protein complexes from the mitochondria of Saccharomyces cerevisiae

Abstract Mitochondria, isolated from Saccharomyces cerevisiae, have been lysed with various detergents and the nucleoid complexes released have been purified on metrizamide gradients. The purification of nucleoids was optimal when the lysis was carried out using Triton X-100 at 30 °C followed by treatment with ribonuclease. The buoyant density of the nucleoids was dependent on the amount of DNA loaded onto the metrizamide gradient and they contained about two and a half times as much protein as DNA. The results obtained suggest that the nucleoids are attached to the inner mitochondrial membrane by RNA and have a regular structure.

Characterization of DNA-protein complexes from the mitochondria of Xenopus laevis oocytes☆

Mitochondrial DNA (mtDNA)-protein complexes (nucleoids) from Xenopus laevis oocytes were purified either on rate-zonal sucrose or isopyknic metrizamide gradients. From electron microscopic studies and staphylococcal nuclease digestion experiments mtDNA appears to be packaged into regular beaded structures. Protein electrophoretic analysis and M banding results show that mtDNA is associated with the membrane structures and also with few specific proteins including one acid-soluble polypeptide of 28 kD.

Separation of T and B lymphocytes from human peripheral blood mononuclear cells using density perturbation methods

The fractionation of sub-populations of peripheral blood mononuclear cells has become an essential routine procedure and some of the main fractionation methods used today are immunomagnetic separations. We describe a less expensive method for the separation of subpopulations of mononuclear cells using density perturbation, which uses the binding of antibody-coated dense polystyrene beads to increase the density of specific sub-populations of cells. By incubating a total mononuclear fraction from human peripheral blood together with antibody-coated beads, in a commercially-available lymphocyte separation medium (Nycoprep 1.077), a depletion of 94.9 +/- 1.68% of the T cells could be obtained by this procedure; a depletion of 69.7 +/- 1.78% of the B cells was also achieved. These results indicate the potential for the separation of different sub-populations of peripheral blood mononuclear cells on the basis of the immunological identity of the surface of cells using density perturbation methods involving antibody-coated dense polystyrene beads.

Buoyant densities of macromolecules, macromolecular complexes, and cell organelles in Nycodenz gradients

Nycodenz is a new nonionic iodinated density gradient medium which has several advantages over metrizamide. Although, overall, biological samples band at similar densities in Nycodenz and metrizamide gradients, a number of significant differences were found. As compared with metrizamide, not only does Nycodenz appear to interact less with proteins but also the buoyant density of chromatin is less affected by the amount loaded onto the gradient. A high degree of resolution is obtainable using Nycodenz gradients; thus, it is possible to separate density-labeled DNA and to subfractionate subcellular membrane fractions.

A new, rapid, one-step method for the isolation of platelets from human blood

We describe a new, rapid method for the isolation of platelets from human blood using a single centrifugation step through Nycodenz, an inert, nontoxic, nonionic medium. As judged by aggregation and nucleotide release studies, the platelets are recovered in high yield and in excellent functional condition.