Differential centrifugation

In biochemistry and cell biology, differential centrifugation (also known as differential velocity centrifugation) is a common procedure used to separate organelles and other sub-cellular particles based on their sedimentation rate. Although often applied in biological analysis, differential centrifugation is a general technique also suitable for crude purification of non-living suspended particles (e.g. nanoparticles, colloidal particles, viruses). In a typical case where differential centrifugation is used to analyze cell-biological phenomena (e.g. organelle distribution), a tissue sample is first lysed to break the cell membranes and release the organelles and cytosol. The lysate is then subjected to repeated centrifugations, where particles that sediment sufficiently quickly at a given centrifugal force for a given time form a compact "pellet" at the bottom of the centrifugation tube. After each centrifugation, the supernatant (non-pelleted solution) is removed from the tube and

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related publications (3)

Related publications

Related people

Related units

Related concepts (10)

Related concepts

Related courses (6)

Related courses

Related lectures (8)

Related lectures

Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells

Daniel Roman Blessing, Nicole Déglon, Vivianne Padrun, Bernard Schneider, Gabriel Vachey, Florian Maria Wurm

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

2019

Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels

Françoise Gisou van der Goot Grunberg

The water permeability of ADH target epithelial cells is believed to be regulated by a cycle of exo-endocytosis of vesicles containing functional water channels. These vesicles were selectively labeled in intact frog urinary bladders with an impermeant fluorescent marker, 6-carboxyfluorescein. Vesicle suspensions containing the labeled endosomes were obtained by homogenization and differential centrifugation of bladder epithelial cells. The osmotic permeability of the endocytic vesicles was measured, using a stopped-flow fluorescence technique, in the absence or in the presence of HgCl2. This permeability was found very high (500 microns/sec) and inhibited by 1 mM HgCl2 (90%), thus confirming the presence of water channels. The labeled endosomes were then separated from the other membrane vesicles by flow cytometry and sorting. Their protein content was analyzed by electrophoresis on ultrathin polyacrylamide gels. Two double bands were found at 71 and 55 kDa as well as a small band at 43 kDa. They respectively correspond to 31, 38 and 10% of the total amount of silver-stained proteins present in the sorted endosomes, while they only represent 2, 4, and less than 1% of the proteins contained in the vesicle suspension, before sorting. These highly enriched proteins (or at least one of them) are likely to be involved in the mechanism of water transport. Associated to their partial purification by differential centrifugation, the sorting of the endosomes by flow cytometry seems a good way to further characterize the water channel.

1992

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

Yiming Li, Françoise Gisou van der Goot Grunberg

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.

2002

Centrifugation

Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed.

Colloid

A colloid is a mixture in which one substance consisting of microscopically dispersed insoluble particles is suspended throughout another substance. Some definitions specify that the particles must

Virus

A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria

CH-210: Biochemistry

Les constituants biochimiques de l'organisme, protéines, glucides, lipides, à la lumière de l'évolution des concepts et des progrès en biologie moléculaire et génétique, sont étudiés.

BIO-105: Cellular biology and biochemistry for engineers

Basic course in biochemistry as well as cellular and molecular biology for non-life science students enrolling at the Master or PhD thesis level from various engineering disciplines. It reviews essential notions necessary for a training in biology-related engineering fields.

BIO-205: Cellular and molecular biology I

The course covers the regulation of gene expression, which translates the information contained in the genome into function, by adjusting the levels and activities of mRNAs and proteins to the needs of specific cells, tissues and environments. A particular emphasis is given on experimental methods.