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PubMed1980

Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll.

Rat mast cells from peritoneal washings were purified by centrifugation on a medium containing silica particles coated with PVP (Percoll). By isopycnic centrifugation on continuous Percoll gradients, the buoyant density of mast cells ranged from 1.09 to 1.17 and was consistently lower in mast cells from younger rats than in mast cells from older rats. In suspensions uncontaminated with blood, the mast cells could be isolated by a simple centrifugation procedure based on density differences (step I). Contaminating red blood cells were removed by a second short centrifugation of velocity gradient type, using a linear Percoll gradient of low density. The mast cell purity was 91-98% and the recovery 63-100%. Purified mast cells reacted normally in a viability test employing fluorescein diacetate, retained amines and heparin, and reacted normally with amine release following incubation with Polymyxin B or antigen (immunised rats). Electron microscopy revealed excellent ultrastructural preservation of the mast cells and infrequent uptake of Percoll particles by endocytosis.

Animals↗
PubMed1994

Induction of low density and up-regulation of CD11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation.

Neutrophils and eosinophils circulating in an activated state are of low density. However, purification procedures such as dextran sedimentation and centrifugation may influence the density and function of cells. In the present study we have evaluated the effect of dextran sedimentation and subsequent centrifugation on the density and CD11b expression of neutrophils and eosinophils. Direct density separation of whole blood resulted in 17.7 +/- 9.0% low density neutrophils (< 1.090 g/ml) and 8.7 +/- 3.5% low density eosinophils (< 1.093 g/ml). Dextran sedimentation at room temperature prior to density separation yielded 57.8 +/- 14.7% low density neutrophils and 43.2 +/- 8.0% of low density eosinophils. Additional centrifugation after dextran sedimentation resulted in an increase of these numbers to 91.7 +/- 3.1 and 69.8 +/- 11.7% respectively. The density shifts were found with hypertonic as well as isotonic Percoll. Furthermore, it was shown that dextran sedimentation resulted in an increased CD11b expression on neutrophils as well as eosinophils. During subsequent washing by centrifugation, a further increase in CD11b expression was observed together with lactoferrin release. The effects of dextran sedimentation on density and CD11b expression were independent of extracellular calcium. These results indicate that dextran sedimentation induces the release of specific granule compartments with subsequent expression of CD11b, resulting in changes in granulocyte density.

Cell Fractionation↗
PubMed1987

Centrifugal ultrafiltration-dialysis for non-protein-bound oestradiol in blood: importance of the support.

The percentage of non-protein-bound oestradiol in serum or other fluid measured by centrifugal ultrafiltration-dialysis was critically dependent on the supporting medium used to collect the ultrafiltrate. When either Whatman No. 1 filter paper discs, Whatman GF/D glass fibre discs or Celite were used, the ratios of 3H-steroid to [14C]glucose on the support were found to change with centrifugation time. This occurred when serum or protein-free filtrates were analysed and was eliminated when silanised Celite was employed as the support. It was also observed that when serum was analysed, the [3H]/[14C] ratio inside the cell increased linearly with centrifugation time and this was ascribed to the small changes in volume which occurred inside the cell as ultrafiltrate accumulated on the support. It was concluded that the correct time to sample the ratio of [3H]/[14C] inside was before centrifugation.

Carbon Radioisotopes↗
PubMed1984

Isopycnic centrifugation of plant viruses in Nycodenz density gradients.

Isopycnic centrifugation of plant viruses in density gradients prepared from a new non-ionic medium (Nycodenz) was investigated. Particle density of luteo-, tymo-, nepo-, cocksfoot mild mosaic, tobamo-, hordei-like, potex- and potyviruses in buffered solutions of Nycodenz ranged between 1.23 and 1.28 g/ml and did not strictly reflect their nucleic acid and protein composition. Isopycnic centrifugation of several partially purified viruses yielded preparations which were pure by electron microscopy and, when used as immunogens in rabbit or hen, gave antisera with low titers of antibodies against host plant antigens, as required for enzyme immunoassays. A preparation of purified potato leafroll virus contained a single dominant protein corresponding to the viral coat protein. It is concluded that isopycnic centrifugation in Nycodenz density gradients is particularly useful for the purification and analysis of plant viruses which are unstable in cesium chloride solutions, such as the luteoviruses, and preferable to zonal centrifugation in sucrose density gradients for the purification of rod-shaped multipartite viruses or viruses which aggregate.

Centrifugation, Isopycnic↗
PubMed1989

A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force.

Determination of the body pattern in Xenopus embryos is known to involve at least six steps. One of these steps can be experimentally simulated by inclining the fertilized egg with respect to gravity or centrifugal force (10-30 g x 4 min, directed 90 degrees to the animal-vegetal axis). In these eggs, the dorsal structures of the body axis form from the side of the egg that was uppermost in the gravitational or centrifugal field. This topography is seen even if the sperm entry point side (the prospective ventral side in control eggs) was uppermost. In addition, conjoined twin embryos form at very high frequencies in response to certain conditions of single or double centrifugation. Cytological analysis shows that the dorsal structures invariably form from the side(s) of the egg away from which vegetal cytoplasm was displaced. This is similar to the situation in the unperturbed egg, where the subcortical cytoplasm of the vegetal hemisphere rotates some 30 degrees relative to the surface, and the dorsal structures form from the side of the egg away from which the subcortical cytoplasm moved. The displacements elicited by centrifugation probably substitute for the normal displacements brought about by the subcortical rotation. These and other data suggest that the subcortical rotation is a crucial step in the process of axis determination. The subcortical rotation is an autonomous activity of the activated egg, and can displace cytoplasm against gravity. I believe that the subcortical rotation will function normally at microgravity, and I expect that overall development and axis polarity at microgravity will be normal. This will be tested in spaceflight.

Animals↗
PubMed1996

Ocular torsion before and after 1 hour centrifugation.

To assess a possible otolith contribution to effects observed following prolonged exposure to hypergravity, we used video oculography to measure ocular torsion during static and dynamic conditions of lateral body tilt (roll) before and after 1 h of centrifugation with a Gx-load of 3 G. Static tilt (from 0 to 57 degrees to either side) showed a 10% decrease in otolith-induced ocular torsion after centrifugation. This implies a reduced gain of the otolith function. The dynamic condition consisted of sinusoidal body roll (frequency 0.25 Hz, amplitude 45 degrees) about an earth horizontal and about an earth vertical axis (respectively, "with" and "without" otolith stimulation). Before centrifugation the gain of the slow component velocity (SCV) was significantly lower "with" otolith stimulation than "without" otolith stimulation. Apparently, the contribution of the otoliths counteracts the ocular torsion response generated by the semicircular canals. Therefore, the observed increase in SCV gain in the condition "with" otolith stimulation after centrifugation, seems in correspondence with the decreased otolith gain in the static condition.

Adult↗
PubMed1979

Hornet building orientation in a vertically rotating centrifuge.

Hornets build combs that are clearly oriented toward the center of gravity and they compensate for any change made in the direction of their cells, provided the gravitational vector remains unchanged. Under natural conditions the first few cells are built by the queen in the spring, following the hibernation period, and building is then continued by the workers. Recently several reports have been published concerning the fact that groups of hornets (V. orientalis workers) may build in the absence of the queen and that such combs resemble those built by the queen. In previous investigations on building orientation, hornets placed in artificial breeding boxes (ABBs) on a horizontal centrifuge were subjected to constant centrifugal and gravitational forces, the resultant of which ranged between 1-1.5 g. Under these conditions young hornets (1-2 days of age) built combs in the direction of the resultant force, while the direction of building by adult hornets (3-7 days of age) was dependent on the presence or absence of a straight roof. Adults placed in the ABBs of rectangular shape built combs the orientation of which was determined by the Earth's gravity alone (perpendicular to the roof) whereas those in spherical ABBs with a convex roof built combs in the direction of the resultant gravitational and centrifugal forces, much the same as combs built by the young hornets. We were, however, also interested in studying the building orientation of hornets placed under conditions of changing resultant forces. The present paper describes comb building by hornets under the alternating gravitational forces of a vertical centrifuge.

Age Factors↗
PubMed2006

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method.

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.

3T3 Cells↗
PubMed1996

Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay.

To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.

Animals↗
PubMed1986

Removal of bacterial contaminants from semen for in vitro fertilization or artificial insemination by the use of buoyant density centrifugation.

Buoyant density centrifugation of semen produces the accumulation of populations of highly motile, morphologically normal spermatozoa in the lowermost 1 ml of Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) density gradients. In addition, the majority of bacteria present in semen are retained in the seminal plasma at the top of the gradients. Of 40 semen samples examined, 37 contained detectable bacteria, but after buoyant density centrifugation, the spermatozoal populations collected from the lowermost 1 ml of the Percoll columns were found to contain few or no bacteria. When preparations were collected using sterile technique (by boring a hole through the bottom of the centrifuge tube), 14 of the 20 preparations were found to be bacteria-free. When preparations were collected by passing a spinal needle from the surface through the seminal plasma to the bottom of the centrifuge tube, the sterility of the final spermatozoa preparations was not maintained, with only 5 of the 20 samples completely free of bacteria. The residual bacterial contamination of the remaining 15 samples was, however, very low (less than 5 colonies after a 48-hour culture period).

Bacteria↗
PubMed1990

Centrifugal affinity chromatography.

A new technique termed centrifugal affinity chromatography (CAC) is presented in this paper. CAC combines a high flow-rate, created by centrifugal force, with the specificity of affinity chromatography. This technique has been used for the purification of human immunoglobulin G. Furthermore this technique has been used to remove human albumin from serum and the effect of centrifugal force, ionic strength and pH has been studied. A test for determining the percentage of glycosylated hemoglobin in hemolysates has also been developed. This test, employing centrifugal chromatography, is more than three times faster than commonly used gravity flow methods.

Centrifugation↗
PubMed1997

Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7.

A sample treatment method based on buoyant density centrifugation which separates bacteria from food, concentrates bacteria and removes PCR inhibitors is described. The method involves a one minute centrifugation of food homogenate layered over a gradient medium (Percoll or BacXtractorTM) in Eppendorf tubes, followed by a single wash step. The small scale of this treatment makes it possible to process many samples in a short time. To evaluate the method beef and minced beef samples, spiked with strains of Escherichia coli O157:H7, were treated and then analysed by PCR aimed at verocytotoxin- (VT1 and VT2) and eae-genes. The detection limits in 1:10 (w/v) beef and minced beef homogenates were 125-250 cfu ml-1 (1250-2500 cfu g-1) and 1000 cfu ml-1 (1 x 10(4) cfu g-1), respectively. The enrichment of spiked samples in buffered peptone water at 37 degrees C for 6 hours before buoyant density centrifugation and PCR, allowed 0.5 cfu g-1 beef and 5 cfu g-1 minced beef to be detected. This combination of enrichment and buoyant density centrifugation was also used for analysis of 43 beef samples from a consignment in which E. coli O157:H7 had been detected, and detected VT-genes in all 43 samples. E. coli O157:H7 was also separated and detected in spiked samples of milk, lettuce, shrimps, and blue cheese at arbitrary concentrations of 3000 cfu ml-1. The present sample preparation method has the potential to be applicable to many other combinations of bacteria and food, and in connection with other detection methods than PCR as well.

Centrifugation, Density Gradient↗
PubMed2000

Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp. in Gouda cheese.

This study examined milk centrifugation, increased salt concentration, and low ripening temperature as potential strategies to prevent late blowing caused by gas-forming Clostridium spp. in Gouda cheese. The survival of clostridia spores in cheese brine and their ability to enter Gouda cheese during brining was also evaluated. Centrifugation (3000 x g for 30 s) of contaminated milk resulted in > 60% spore reduction, with increased spore reduction at greater centrifugal forces. Low levels of C. tyrobutyricum and C. sporogenes spores survived in saturated (23%, w/v) brine with 2% (v/v) added whey at 15 degrees C for 63 days, while C. beijerinckii and C. butyricum spores were not detectable on days 4 and 35, respectively. Spores of C. tyrobutyricum in brine infiltrated Gouda cheese during 2 h of brining at 13 degrees C resulted in production of small gas holes during ripening. In Gouda cheese slurry stored at 13 degrees C, three C. tyrobutyricum strains plus one of three C. sporogenes strains germinated in the slurry with no added salt. Of three C. tyrobutyricum strains stored at 13 degrees C in slurries with higher water-phase salt concentrations of 2.4 and 3.6%, two strains and one strain germinated, respectively. No germination of spores was detected in any cheese slurry stored at 5 or 8 degrees C. Milk centrifugation, increased percent water-phase salt, absence of spores in brine, and decreased ripening temperature are all potentially important measures against gas production by Clostridium spp. in Gouda cheese.

Animals↗
PubMed2003

Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation.

BACKGROUND: Human cytomegalovirus (HCMV) is reactivated in nearly every seropositive breastfeeding mother during lactation [Lancet 357 (2001) 513]. Conventional tissue culture (TC) and low-speed centrifugation-enhanced microtiter culture methods are not able to detect HCMV from milk during all stages of lactation. OBJECTIVES: Development of a sensitive and quantitative microculture technique to describe the dynamics of HCMV reactivation in different milk compartments during lactation. STUDY DESIGN: Milk samples were collected longitudinally from seropositive breastfeeding mothers of preterm infants. Native milk samples were separated into fraction 1 (aqueous extract of milk fat), fraction 2 (cell and fat free milk whey) and fraction 3 (milk cells). Each of these fractions was screened qualitatively (TC, nPCR, pp67 late mRNA) and quantitatively (high-speed centrifugation-based microculture, quantitative PCR). RESULTS: Prior to low-speed centrifugation-enhanced inoculation, virus particles were concentrated by high-speed centrifugation (60 min at 50,000 x g, 4 degrees C). Using fraction 2 we were able to describe the dynamics of viral reactivation during lactation. We present the course of the quantitative virolactia and DNAlactia and qualitative detection of HCMV pp67 late mRNA in milk whey of four mothers (three transmitters and one non-transmitter). In all these cases virolactia described an unimodal and self limited course. Peak levels of virolactia for transmitters (T1: day 44; T2: day 43; T3: day 50) were closely related the onset of viruria of the corresponding preterm infants (U1: day 39; U2a/U2b: day 44/57; U3: day 60). The courses of viral load coincidence with the courses of DNA load. CONCLUSIONS: We present a rapid and highly sensitive microculture method for the quantification of cell free HCMV from milk whey and aqueous extracts from milk fat. Viral reactivation during lactation describes an unimodal course. Our findings have strong implications for quality control of any virus inactivation procedure.

Breast Feeding↗
PubMed1981

Pressure-induced inactivation of sarcoplasmic reticulum adenosine triphosphatase during high-speed centrifugation.

Sarcoplasmic reticulum vesicles were found to be highly sensitive to high-speed centrifugation in metal-deprived mediums at low temperature (4 degrees C). The irreversible modifications induced were easily detected from observation of the environment-sensitive spectrum of an iodoacetamide spin-label bound to the ATPase. Centrifugation also resulted in vesicle aggregation and inhibition of calcium transport, ATPase activity, and phosphoenzyme formation. These denaturation-like phenomena were prevented in the presence of sucrose, or by nucleotide binding, or, again, by cation binding to the ATPase high-affinity calcium binding sites and were only present when centrifugation was performed at low temperature. The crucial parameter during this process was found to be the hydrostatic pressure which developed in the centrifuge tube. SR vesicles exposed to 800 bars in a pressure bomb displayed the same features. It is suggested that irreversible denaturation takes place after one or both of the two following well-documented effects of pressure: a rise in the lipid order/disorder transition temperature or dissociation of the oligomeric structure of the calcium pump.

Adenosine Triphosphatases↗
PubMedDate not supplied

Impact of process conditions on the centrifugal recovery of a disabled herpes simplex virus.

Despite continuous improvements in culturing and recovery techniques, high-titer stocks of purified disabled herpes simplex virus type-1 (HSV-1 DIS) vector for drug discovery and use in preclinical and clinical trials are currently difficult to achieve. Efforts to improve their centrifugal recovery have been addressed in this paper. The operation of a swing-out centrifuge rotor was assessed, and its operational conditions were defined for the recovery of viable HSV-1 DIS. 80% virus recovery was achieved after 90 min at 26000g. The 20% loss of virus was attributed to damage to the viral envelope by overcompaction of the pellet and impaction with the base of the centrifuge tube. Virus recovery was increased by a further 10% by using a fixed-angle centrifuge rotor operating at 26000g. Plaque assays of recovered HSV-1 DIS gave values on the order of 10(6) pfu/mL, compared to values typically above 10(9) pfu/mL obtained for the replication-competent HSV-1 viron.

Animals↗
PubMed1997

The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer.

Pretreatment of retroviral supernatants with the cationic liposomes DOTMA-DOPE (Lipofectin), DC-Chol-DOPE and DOSPA-DOPE (Lipofectamine) was found to enhance static transductions of TF-1 target cells. The relative effectiveness at increasing transduction efficiencies (TE) was: DOSPA > DC-Chol > DOTMA, resulting in average increases over nontreated controls of 11.9-, 6.2- and 1.2-fold, respectively. This pretreatment was found to be synergistic when combined with centrifugation, having the same order of effectiveness, and resulting in 57-, 35- and 27-fold increases over nontreated controls. For Lipofectamine and DC-Chol-DOPE liposomes, the combined approach yielded 2.2- and 1.3-fold increases over untreated centrifuged samples. Individual colonies picked from colony-forming unit granulocyte-macrophage assays of infected CD34+ cells were screened for the presence of the transgene by polymerase chain reaction (PCR). Colonies from cells infected using centrifugation were positive 27% of the time, while the combined approach had positive colonies 31 and 50% of the time for DC-Chol and Lipofectamine, respectively. The addition of protamine sulfate to the liposome-supernatant mixture during pretreatment was found to be inhibitory. With increasing centrifugal force, the TE of cells infected with Lipofectamine pretreated and untreated supernatants increased proportionally. However, the TE of the cells infected with the pretreated supernatants was significantly higher than the TE of the cells infected with untreated supernatants at all points examined. The increase in TE associated with liposomal pretreatment of retroviral supernatants was not shown to be attributed to a nonreceptor-mediated pathway for viral entry into the cell.

Antigens, CD34↗
PubMed1999

Improving electrotransfection efficiency by post-pulse centrifugation.

We have demonstrated that the viability of electrotransfected adherent CHO and suspended NK-L, K-562, L1210 and MC2 cells is improved if pelleting by centrifugation is performed immediately after pulsing. The protection effect on cell viability is cell line- and pellet thickness-dependent. For forming CHO cell pellets, centrifugation force (300-13,000 g) and duration are not crucial; about five to 10 cell layers in the pellet provide the optimal protection effect. NK-L, K-562, L1210 and MC2 cell pellets are optimally formed by centrifugation at 13,000 g in an Eppendorf desktop centrifuge. Pelleting improves the cell viability over the whole range of the NK-L, K-562, L1210 and MC2 cell concentrations studied. When this pelleting method is applied to load CHO cells with FITC-dextran (41,000 MW), not only is the success rate close to 100%, but the growth rate is similar to the control, which is far better than the conventional electroporation method. Furthermore, the transfection efficiency of the five cell lines in pellet is significantly higher than that in suspension.

Animals↗