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PubMed1997

Sperm count in ejaculates and after sperm selection with discontinuous percoll gradient centrifugation technique, as a prognostic index of IVF outcome.

Success of IVF with low sperm count will depend on the retrieval of the maximum number of normal motile sperms, and this makes the selection of an appropriate technique critical. Various in vitro methods have been developed for selecting human sperm cells. Sperm selection using percoll gradient has been reported to yield upto 60% motile and morphologically normal spermatozoa from a normal semen sample. In this study, we have attempted to determine a possible relationship between sperm count in ejaculates before and after selection with percoll gradient centrifugation on one hand, and fertilization, cleavage and pregnancy rates in an IVF program on the other. With increased sperm count at the time of IVF treatment using selected sperm, we observed higher fertilization, cleavage and gestational rates. In conclusion, sperm concentration before and after selection with percoll may be considered a prognostic parameter for the determination of fertilization potential and pregnancy rates in an IVF program.

Cell Division↗
PubMed1985

Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by Percoll density gradient centrifugation.

A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic beta-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells.

Animals↗
PubMed1981

Erythrocyte osmotic fragility in various liver diseases--application of coil planet centrifuge system.

A change in erythrocyte osmotic fragility was observed on various liver diseases by means of the coil planet centrifuge (CPC) system, and the relationship between changes in it and in serum lipids was studied. According to the CPC classification of hemolytic patterns of L, M, T and R, the frequency of appearance of T and R increased in liver cirrhosis and primary hepatoma. Hemolytic start and end points both changed considerably in primary hepatoma, acute hepatitis and liver cirrhosis. Change of hemolytic end point which shifted to the hypotonic side is more prominent than that of hemolytic start point. The hemolytic end point showed an inverse correlation to serum alkaline phosphatase and LAP, and correlation to pseudocholinesterase and albumin. Among the relations of red cell fragility and lipids of the lipoprotein fractions, free cholesterol and the ratio of free cholesterol to phospholipid in high density lipoprotein were both in remarkable inverse correlation to the hemolytic end point. Free cholesterol in high density lipoprotein was concluded one of the most important determinants of erythrocyte osmotic fragility.

Acute Disease↗
PubMed1999

Radiotracer analysis of cadmium speciation in soil solutions using electrophoresis, dialysis, centrifugation, and ultrafiltration.

Speciation of carrier-free 109Cd, added in cationic form to pre-filtered extracts obtained by leaching forest soil samples with distilled water, was analyzed using electrophoresis, dialysis, centrifugation, and ultrafiltration. Rapid establishment of isotopic equilibrium between the added 109Cd and stable cadmium present in the extracts was observed. All the data obtained indicated that 109Cd and also stable cadmium were present in the analyzed extracts in the form of neutral or negatively charged organic complexes or small colloids. The results of electrophoresis enabled the characterization, at least semiquantitative, of the abundance and electrophoretic mobility of the forms present. The incomplete dialysis of 109Cd from the soil extracts through cellophane membrane against water proved the presence of organic associates with molecular weights higher than 10(4). Dialysis against the same, but unlabeled extract was always complete, indicating the reversible (labile) nature of the organic forms of cadmium. Assessment of the stability constants of the organic forms using a simple discrete two-site model suggested that humate and/or fulvate complexes of cadmium were formed.

Cadmium↗
PubMed1980

Synchronization of 9L rat brain tumor cells by centrifugal elutriation.

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.

Animals↗
PubMed1983

Theory and practice of centrifugal elutriation (CE). Factors influencing the separation of human blood cells.

Centrifugal elutriation (CE) is currently a widely used preparative cell separation technique. In order to optimize the separation of cells that show only small differences in sedimentation velocity, several conditions that might influence the resolution capacity, such as rotor speed, counterflow, jetstream, cell load, density, and viscosity of the elutriation medium, were analyzed. Experiments carried out with human red blood cells (rbc) indicated that selective losses of rbc from the rotor caused by the jetstream, could be prevented if the separations were carried out at high rotor speeds, as predicted by the theory. In addition, high cell loads (5 X 10(8) rbc) resulted in better separations than low cell loads (5 X 10(7) rbc). Human monocytes were separated into subpopulations that differed only about 0.003 g/mL in density, but have virtually the same size. The separation was carried out either by increasing the density or viscosity of the elutriation medium or by decreasing the rotor speed. In all cases similar results were obtained. These results indicated that under optimal conditions CE can be applied for the separation of cells that differ only slightly in sedimentation velocity.

Blood Cells↗
PubMed2002

Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli.

Cultures of Escherichia coli grown in space reached a 25% higher average final cell population than those in comparably matched ground controls (p<0.05). However, both groups consumed the same quantity of glucose, which suggests that space flight not only stimulated bacterial growth as has been previously reported, but also resulted in a 25% more efficient utilization of the available nutrients. Supporting experiments performed in "simulated weightlessness" under clinorotation produced similar trends of increased growth and efficiency, but to a lesser extent in absolute values. These experiments resulted in increases of 12% and 9% in average final cell population (p<0.05), while the efficiency of substrate utilization improved by 6% and 9% relative to static controls (p=0.12 and p<0.05, respectively). In contrast, hypergravity, produced by centrifugation, predictably resulted in the opposite effect--a decrease of 33% to 40% in final cell numbers with corresponding 29% to 40% lower net growth efficiencies (p<0.01). Collectively, these findings support the hypothesis that the increased bacterial growth observed in weightlessness is a result of reduced extracellular mass transport that occurs in the absence of sedimentation and buoyancy-driven convection, which consequently also improves substrate utilization efficiency in suspended cultures.

Biological Transport↗
PubMed1992

Centrifugal separation of carcinoma or atypical cells in voided urine.

A simple density gradient method was used to separate atypical and cancer cells from non-cancer cells in voided urine from patients with transitional cell atypia (moderate and grave atypia) and bladder cancer (squamous cell carcinoma and transitional cell carcinoma). Prior to cell separation, the Saccomanno preserved cells were dispersed by homogenization. After cell separation (5 min x 1400 rpm), atypical and cancer cells were enriched up to 20-fold. Also, most of the leucocytes (68-98%) and squamous cells (47-82%) were absent from density gradient specimen fractions containing the largest percentages of atypical and cancer cells. Peak purity ranges of atypical or cancer cells from different sample classes showed a large degree of overlap. This permitted the pooling of density gradient fractions enriched for atypical or cancer cells, thus increasing the efficiency of the method. Also, following centrifugation, the Papanicolaou-stained specimen fractions showed less background staining than the unprocessed controls, and the cells retained diagnostic morphologic features. We infer that this method may be a useful, low-cost approach for the morphologic study of developing cancers, not only from the urinary bladder, but also from the respiratory tract.

Carcinoma, Squamous Cell↗
PubMed2001

A simple centrifugation method for harvesting myeloblasts.

Myeloblast-rich samples, required for investigation of myeloid malignancies, can be obtained only during the untreated stage of leukemia. Existing methods for myeloblast enrichment have various prerequisites that limit their application. In this new method, a mixture of peripheral blood (Mixed PB) from an acute myeloid leukemia (AML) patient and from a healthy control containing 5% myeloblasts was subjected to density gradient centrifugation using a 14.5% metrizamide solution. Both high purity (86.3% +/- 1.5%) and high recovery of viable myeloblasts were achieved. Close to 100-fold blast enrichment, even from Mixed PB containing only 0.15% myeloblasts, was achieved. Similarly, this method highly enriched myeloblasts from unprocessed samples, including marrow cells, from patients with AML, myelodysplastic syndromes, and chronic myeloid leukemia (purity: 2.7% +/- 2.0% before separation, 56.6% +/- 28.3% after separation) (n = 22). The enriched blasts were suitable for various analyses, eg, flow cytometry, immunocytochemistry, cytochemistry, fluorescence in situ hybridization, and gene analysis.

Acute Disease↗
PubMed2004

[Epitheliotrophic capacity of serum and plasma eyedrops. Influence of centrifugation].

BACKGROUND: Serum eyedrops are a new modality for the treatment of ocular surface disorders. We examined the influence of the preparation of blood products in a cell culture model and compared it with plasma. MATERIAL AND METHODS: Serum and plasma were obtained from full blood of ten healthy volunteers and centrifuged at 500 and 3000 G. EGF, PDGF, TGF-beta1, fibronectin, and vitamin A were quantified by means of ELISA and HPLC. Cultures of human corneal epithelial cells were incubated with the four blood products in dose-response experiments and the intracellular ATP quantified. RESULTS: EGF, PDGF, and vitamin A were present in serum in significantly higher concentrations than in plasma. The concentration of fibronectin was not influenced by the preparation. Support of proliferation was best by 25% platelet-poor serum. Serum supported the differentiation and migration of epithelial cells better than plasma. CONCLUSION: The biochemical character of serum eyedrops is determined by the parameters chosen to produce the blood product. Plasma does not seem to offer an epitheliotrophic capacity equivalent to serum eyedrops. Their production should be optimized before any meaningful randomized controlled clinical trial can be attempted.

Adenosine Triphosphate↗
PubMed2004

Graviperception in growth inhibition of plant shoots under hypergravity conditions produced by centrifugation is independent of that in gravitropism and may involve mechanoreceptors.

Hypergravity caused by centrifugation inhibits elongation growth of shoots by decreasing the cell wall extensibility via suppression of xyloglucan breakdown as well as by the thickening of cell walls. The mechanism of graviperception in hypergravity-induced growth inhibition was investigated in Arabidopsis [A. thaliana (L.) Heynh.] hypocotyls and azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. Hypergravity caused growth suppression in both sgr1-1 and pgm1, which are Arabidopsis mutants deprived of gravitropism, as in wild-type plants, suggesting that the graviperception in hypergravity-induced growth inhibition of shoots is independent of that in gravitropism. Hypergravity had no effects on growth of azuki bean epicotyls or Arabidopsis hypocotyls in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. Moreover, lanthanum or gadolinium at the same concentration had no influence on gravitropism of azuki bean epicotyls and Arabidopsis hypocotyls. Hypergravity had no effects on cell wall extensibility and affected neither xyloglucan metabolism nor the thickness of cell walls in the lanthanum- or gadolinium-treated azuki bean epicotyls. Lanthanum or gadolinium inhibited the hypergravity-induced increase in the pH of the apoplastic fluid in the epicotyls, which is involved in the processes of the suppression of xyloglucan breakdown due to hypergravity. These findings suggest that plants perceive the hypergravity stimuli by mechanoreceptors in the plasma membrane, and utilize the perceived signal to regulate the growth rate of their shoots.

Arabidopsis↗
PubMed1997

Isolation of coccidian enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of cats by Percoll density-gradient centrifugation.

A method for isolation of enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of experimentally infected cats was developed using Percoll density-gradient centrifugation. Gamonts and merozoites were obtained essentially free of host-cell debris. A recovery rate of nearly 30% of the parasites in the original preparations was obtained by this method. Merozoites were separated from gamonts by filtration through a 3-micron polycarbonate filter.

Animals↗
PubMed2004

Fully integrated L-phenylalanine separation and concentration using reactive-extraction with liquid-liquid centrifuges in a fed-batch process with E. coli.

A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E. coli F-4/pF81 is presented. The strain was rationally constructed for the production of the aromatic amino acid. Glucose and tyrosine control is used. A reactive extraction system consisting of kerosene, the cation-selective carrier D(2)EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate. Permeate is drained off from the bioreactor bypass. Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28% relative glucose/L-phe yield increase is observed. Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.

Centrifugation, Density Gradient↗
PubMed1983

Surface labeling of thyrocytes isolated by a new method combining enzymatic digestion and percoll gradient centrifugation.

Thyrocytes were isolated from porcine thyroid glands using a new method entailing two-step collagenase digestion and Percoll gradient centrifugation. Good yield and a high percentage of viable thyrocytes free from other contaminating cells was achieved. Proteins present on the surface of thyroid cell plasma membranes were then specifically labeled using Iodogen and 125I-. Membrane lysates were separated by electrophoresis on 10% polyacrylamide gel, under reducing and nonreducing conditions, followed by autoradiography. When the gels were stained with silver nitrate some 30 bands were visualized in both the presence and absence of reductant. Only 9 bands were found to be labeled under nonreducing conditions and 12 in the presence of reductant. Two bands involved in the thyrotropin receptor structure--Mr = 66,000 and Mr = 70,000, respectively--were visualized in the absence of reductant. Upon reduction the Mr 66,000 band was retained and a new band (Mr = 33,000) was seen. The mild enzymatic treatment used in isolating thyrocytes and the lack of contamination with other cells allowed the consistent labeling of exposed plasma membrane components by the Iodogen method such that the orientation of thyrotropin receptor components in the plasma membrane could be deduced.

Animals↗
PubMed1984

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique.

An in vivo-in vitro alkaline DNA unwinding assay for single-strand breaks and/or alkali-labile lesions in hepatic DNA is described. The assay involves isolation of hepatic nuclei from mice, alkaline denaturation and unwinding of hepatic DNA, separation of single- and double-stranded DNA by hydroxylapatite batch chromatography, and quantitation of DNA in chromatographic fractions by a fluorometric assay. The method allows for the sensitive detection of hepatic DNA damage following in vivo administration of xenobiotics. Using this procedure, DNA fragmentation was demonstrated in alkali after administration of as little as 0.5 mg/kg of N-nitrosodimethylamine (DMN) to male B6C3F1 mice. A comparison of this technique with a similar alkaline sucrose density gradient centrifugation assay demonstrated comparable limits of sensitivity for the two procedures.

Alkalies↗
PubMed1985

Isolation of enzymes from polyacrylamide disk gels by a centrifugal homogenization method.

A sliced segment of polyacrylamide gel was quickly homogenized without any loss of gel pieces. The gel segment was placed on a disposable pipet tip, which was packed with a small amount of lumped copper wires and held in a microfuge tube. The gel was homogenized by centrifugation for 15 s at 15,000 g at 0 to 4 degrees C. Almost 70% of endodextranase activity could be recovered from homogenized gel within 30 min at 4 degrees C, whereas only 20% of activity was eluted from gel slices. If necessary, copper wire could be replaced by fine stainless-steel wire or by the nylon string used in fishing lines. Proteins could also be recovered from the homogenized gel by charging electric current for 1 h at 4 degrees C.

Centrifugation↗
PubMed1985

Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation.

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.

Cell Fractionation↗
PubMed1985

Preparative isolation of peroxisomes from liver and kidney using metrizamide density gradient centrifugation in a vertical rotor.

A method for the preparative isolation of peroxisomes from the livers of rat, guinea pig, and mouse, and also from rat kidney is described. The light mitochondrial fraction, i.e., particles sedimenting between 33,000 and 250,000g-min, or the postnuclear supernatant of liver or kidney, is subjected to a 20-50% Metrizamide density gradient ultracentrifugation in a vertical rotor. After centrifugation, the peroxisomes (marker enzyme catalase and dihydroxyacetone phosphate acyltransferase) sedimented as a band near the bottom of the tube (rho = 1.22 g/ml). From the distribution of different marker enzymes and also from the morphometric examinations, it was demonstrated that the isolated peroxisomes are not contaminated with lysosomes, mitochondria, or microsomes.

Animals↗