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At least 829 records · Page 46Linked to original sources
PubMed2003

Centrifugal partition chromatography: a survey of its history, and our recent advances in the field.

Centrifugal partition chromatography (CPC) was introduced in 1982 by Sanki Eng. And for almost 20 years this company remained the only one manufacturing such an apparatus. A CPC instrument or a CPC column is a series of channels linked in cascade by ducts and aligned in cartridges or disks in a circle around a rotor; setting the rotor in motion submits this assembly to a constant centrifugal field. The originality of CPC is that it uses any biphasic liquid-liquid system as mobile and stationary phases. The United States branch of Sanki contributed greatly to the worldwide acceptance of the technique. Recent works performed in the Netherlands and in France have, by means of visualization of flow-patterns in CPC channels, contributed to a better knowledge of hydrodynamics and mass transfer phenomena. Nowadays research in our laboratory focuses on speediness and scale-up of the technique.

Centrifugation↗
PubMed1998

Ligand binding to macromolecules or micelles: use of centrifugal ultrafiltration to measure low-affinity binding.

We describe a method for estimating ligand binding to a macromolecular sample under conditions where this binding is of low affinity and must be measured under equilibrium conditions, without removal of the unbound ligand. The method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the difference between the (total) ligand in the concentrated sample and the (free) ligand in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to separate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of bound ligand becomes significant, even under low-affinity binding conditions). We applied this technique, using Centricon 10 (Amicon) devices, to several cases (soluble proteins, intact membranes, detergent-solubilized proteins, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose filters (nonspecific ligand adsorption and protein denaturation).

Adenosine Triphosphate↗
PubMed2000

Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation.

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) through a long separation channel under a centrifugal force field. Protein samples are exposed to a gradually increasing AS concentration and precipitated along the channel. Then, chromatographic elution is initiated by gradually decreasing the AS concentration in the gradient which causes the proteins to repeat dissolution and precipitation through the channel. Consequently, they are eluted out in the order of their solubility in the AS solution. The separation column consists of a pair of disks equipped with mutually mirror-imaged spiral grooves. A dialysis membrane is sandwiched between the disks to form two identical channels partitioned by the membrane. The disk assembly is mounted on the sealless continuous-flow centrifuge. When a concentrated AS solution is eluted through one channel and water through the other channel in an opposite direction, an exponential AS gradient is formed through the water channel. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of gradient, and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatant and an affinity separation of recombinant ketosteroid isomerase from a crude Escherichia coli lysate.

Ammonium Sulfate↗
PubMed2002

Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation.

We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).

Animals↗
PubMed1997

Cryopreservation of mature bovine oocytes following centrifugation treatment.

In vitro matured bovine oocytes were frozen slowly in 1.6 M 1,2-propanediol following centrifugation treatment for polarization of lipid droplets in the cytoplasm. After thawing, the survival of the oocytes was assessed morphologically and also by in vitro fertilization and culture. The polarization of cytoplasmic lipid droplets had a negative effect on the survival of frozen-thawed oocytes. Thus, this treatment did not improve the frequency of normal fertilization and development to blastocysts, compared with that of frozen control oocytes. However, the frequency of polyspermy and activation of lipid-polarized oocytes that survived after freezing-thawing and subsequent in vitro fertilization tended to be less than those of surviving control oocytes. In addition, the effect of centrifugation treatment was to produce a small but significant increase in the cleavage rate of oocytes that survived after freezing-thawing and the development rates to blastocysts of surviving lipid-polarized oocytes tended to increase, compared with those of surviving control oocytes. These results suggest that the freezing tolerance of the spindle and other organelles of in vitro matured bovine oocytes is associated with lipid droplets and may be improved by the polarization of cytoplasmic lipid droplets before cryopreservation.

Animals↗
PubMed1993

Growth-regulatory properties of G1 cells synchronized by centrifugal elutriation.

In this study we investigated whether early G1 cells can be collected by the method of centrifugal elutriation, and in particular whether the stringent growth regulatory properties characteristic of early G1 cells really are preserved in elutriated cells. We compared the cell cycle kinetics of elutriated Swiss 3T3 cells with unperturbed cells which had been analyzed by time-lapse video recording. Our data show that centrifugal elutriation yields a good synchronization of early G1 cells. However, from a strict cell cycle kinetic point of view the elutriated cells preserve their growth regulatory properties to a variable extent. This fact was explained by the finding that the early G1 cells tend to undergo a temporary G0 arrest as a result of the elutriation procedures.

3T3 Cells↗
PubMed1985

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips.

The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11-45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.

Animals↗
PubMed1984

Separation of immunoreactive lymphocytes from human pluripotent stem cells (CFU-GEMM) by means of counterflow centrifugation.

Counterflow centrifugation with continuous monitoring of the output for cell number and cell scatter was used to separate low density (d less than 1.070 g/ml) human bone marrow cells in two fractions: one containing the majority of small size lymphocytes and the other the majority of the larger sized committed progenitor cells. The recovery of the pluripotent stem cells (CFU-GEMM) in the large cell fraction was complete. The mitogenic reactivity of this putative stem cell fraction had decreased to 6% and 11%, of the original value as measured with phytohemagglutinin stimulation and one way mixed lymphocytic culture respectively. Counterflow centrifugation offers a physical separation technique, by which the majority of the immunoreactive cells can be separated from the pluripotent hematopoietic stem cells.

Bone Marrow Cells↗
PubMed1988

Purification of rat cutaneous mast cells with Percoll density centrifugation.

The skin is the major site on anaphylaxis, and cutaneous mast cells have an important role in its reactions. The isolation and purification of rat cutaneous mast cells are described here. Rat abdominal skin was digested with collagenase and hyaluronidase, and centrifuged with Percoll. The buoyant density of cutaneous mast cells was high, and relatively pure mast cells were obtained. The purity of cutaneous mast cells was 74% +/- 2.4% before and 50.0% +/- 6.4% after Percoll density centrifugation; peritoneal mast cells revealed 5.8% +/- 1.3% purity before and 61.0% +/-10.6% purity after the same procedure. The isolated cutaneous cells released 21.3% +/- 3.8% histamine and the peritoneal mast cells released 55.5% +/- 3.8% histamine upon stimulation with 10 micrograms/ml compound 48/80. These findings suggest that there are functional subsets of connective tissue mast cells.

Animals↗
PubMed1987

Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation.

Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll density centrifugation. Isolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle- and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flattened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.

Actins↗
PubMed1984

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry.

A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.

Animals↗
PubMed1979

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation.

Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.

Adrenal Cortex↗
PubMed1988

Comparison of the in vitro fertilization rate by human sperm capacitated by multiple-tube swim-up and Percoll gradient centrifugation.

Two sperm preparation methods, a multiple-tube swim-up and Percoll-gradient centrifugation, were employed in our human in vitro fertilization program. The fertilization rate of these two sperm preparation methods was compared when they were employed in semen samples of less than 60 million motile sperm/ml. The results described here suggest that both of these methods gave a similar fertilization rate in these semen samples, i.e., 72 +/- 8% for the Percoll-gradient centrifugation method and 66 +/- 8% for the multiple-tube swim-up method.

Centrifugation, Density Gradient↗
PubMed1975

Use of iodinated organic compounds for the density gradient centrifugation of viruses.

Use of Urografin and Conray for the equilibrium centrifugation of viruses is described. These pharmaceuticals, which consist of iodinated arylic compounds, reach densities of 1.6 g/cm3 and have low intrinsic viscosities. Poliovirus, Newcastle disease virus, and lymphocytic choriomeningitis virus were centrifuged to equilibrium in gradients made of these substances. Viral infectivities were not measurably affected, which is especially noteworthy in the case of the very labile lymphocytic choriomeningitis virus. Buoyant densities were found to be significantly lower than densities obtained with gradients made of CsCl and sucrose.

Centrifugation, Density Gradient↗
PubMed1995

Centrifugal and roller pumps--are there differences in coagulation and fibrinolysis during and after cardiopulmonary bypass?

A number of hemostatic parameters reflecting the activation of coagulation and fibrinolysis were investigated in a prospective study of 24 patients undergoing cardiopulmonary bypass (CPB) during heart surgery. The patients were randomized to a group in which either a roller (group 1) or a centrifugal pump (group 2) was used. Blood samples were taken preoperatively, at the onset of and every 20 min during CPB, after the administration of protamine, and 4, 20, 44, and 68 h postoperatively. The groups did not differ significantly in hematocrit, fibrinogen, factor XIII, and antithrombin III. Significant differences in favor of group 2 during and after CPB were found in prothrombin fragment F1 + 2, plasmin-antiplasmin complex (PAP), thrombin-antithrombin complex (TAT), and D-dimer (F1 + 2 P < 0.01 after 80-min CPB, PAP P < 0.005 after 40-min CPB, TAT and D-dimer P < 0.05 after 100-min CPB, D-dimer and PAP P < 0.05 after protamine administration, TAT and F1 + 2 4 h after CPB). These findings indicate the activation of fibrinolysis preceding thrombin generation during cardiopulmonary bypass. In addition, we conclude that centrifugal blood pumping is beneficial in avoiding excessive activation of both coagulation and fibrinolysis.

Aged↗
PubMed1991

Comparison of MRC-5 and U-373MG astrocytoma cells for detection of cytomegalovirus in shell vial centrifugation cultures.

U-373MG astrocytoma cells are susceptible to human cytomegalovirus (CMV) infection and offer the advantage of a continuous cell line for clinical laboratory use. U-373MG to MRC-5 cells for detection of CMV by centrifugation culture were therefore compared. At 20 h, 10 (6.1%) versus 12 (7.4%) of 163 clinical specimens were positive for CMV, and at 40 h, 12 (7.4%) versus 17 (10.4%) were positive in U-373MG and MRC-5 cells, respectively. Substantial toxicity was found in U-373MG cells (84%) when inoculated with blood specimens. For detection of CMV in centrifugation culture, MRC-5 cells are superior due both to higher sensitivity and lesser toxicity.

Astrocytoma↗
PubMed1991

Detection of Chlamydia trachomatis by direct immunofluorescence improved by centrifugation of specimens.

During a study of women with laparoscopically investigated pelvic pain, genital tract specimens were examined for Chlamydia trachomatis using a direct fluorescent antibody (DFA) technique (MicroTrak, Syva) and culture. Some smears, particularly those from the upper genital tract, contained an inadequate number of cells when examined by the DFA technique and many cell monolayers were destroyed by the specimens. To obtain results for such samples, or to confirm the original DFA result, additional specimens which had been frozen at -70 degrees C or in liquid nitrogen were centrifuged at high speed and the resulting deposit examined by the DFA technique. By this means, 32 negative results were confirmed for specimens from 10 chlamydia-negative patients with pelvic inflammatory disease or with high chlamydial antibody titres, and 26 negative results were confirmed for 19 patients who were positive at other sites. In addition, three chlamydia-positive and six chlamydia-negative results were obtained for sites where the original smear for DFA testing had been inadequate (few epithelial cells) and six specimens that were negative originally were found to be positive. Thus, of 73 specimens that were either inadequate or negative by DFA testing originally, 9 (12%) were positive by DFA testing after centrifugation.

Centrifugation↗
PubMed1988

Erythrocyte contamination of leukocyte populations following density-gradient centrifugation results in artificially high levels of human leukocyte HMG-CoA reductase activity.

When measuring human leukocyte HMG-CoA reductase activity, special care must be taken to prevent erythrocyte contamination of the leukocyte layer during isopycnic centrifugation. Contamination during leukocyte isolation and subsequent erythrocyte lysis during NH4Cl treatment results in increased leukocyte microsomal HMG-CoA reductase activity. Increased enzyme activity is not due to enzyme dephosphorylation, thiol-disulfide reduction or increased enzyme protein concentration. Leukocyte populations containing granulocytes appear to be most sensitive. Prevention of erythrocyte contamination during isopycnic centrifugation should aid in accurate measurement of human leukocyte HMG-CoA reductase activity.

Blood Proteins↗