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PubMed1980

Utility of density gradient centrifugation for fractionation of leukemic bone marrow.

Comparison of the distribution pattern of leukemic and normal bone marrow cells, obtained by density gradient centrifugation, may give information about the location of the blockade in the differentiation pathway from stem cell to mature leukocyte, as is supposed to exist in acute leukemia. As a control group 12 children with acute lymphoblastic leukemia (ALL), in complete remission and after cessation of all antileukemic therapy, were studied. By centrifugation over a discontinuous bovine serum albumin (BSA) density gradient, good reproducibility of the partition of the bone marrow cell population was obtained, with a peak in the more dense region. The cells capable of incorporation of [3H] thymidine in vitro were present predominantly in the lower density range. In 9 patients with lympho- or myeloproliferative malignancies (3 with o-cell ALL, 2 with T-cell malignant lymphoma, 1 with B-cell malignant lymphoma, 3 with various malignancies of the myeloid-monocytoid series) similar fractionation on the BSA gradient revealed a different cell partition pattern as compared with the control group. The peak of the distribution curve was located in the less dense region. Highest [3H] thymidine incorporation values were measured in vitro with cells from the lower density region. At last the importance of purely practical considerations in the choice of material for density gradient fractionation is stressed. Application of density gradient centrifugation methods could probably be helpful in attributing to cells from acute leukemia a place in the normal differentiation pathway.

Bone Marrow↗
PubMed1987

The distribution of organelles in mammalian oocytes following centrifugation prior to injection of foreign DNA.

The centrifugation of oocytes from the domestic species is a necessary prerequisite to allow visualization of nuclei for the introduction of foreign DNA. This improvement in visibility which has allowed the production of transgenic animals is accompanied by a clear stratification of the organelles. In immature oocytes from the sheep, pig, and cow, four distinct zones are formed. These comprise, lipid, membrane-bound vesicles, organelle-free cytoplasm, and mitochondria. In mature ovine oocytes, a fifth zone of smooth endoplasmic reticulum (SER) beneath that of the vesicles is formed. This SER is produced at discrete locations within the untreated cell. The potential for removal of these fractions has implications for relating patterns of protein synthesis with particular structural components. In intact oocytes, the cortical granules are located in a peripheral position beneath the plasma membrane. However, even after subjection to high centrifugal force (65,000g for 60 min), they maintain their original location. However, treatment with the cytoskeletal inhibitors, nocodozole and cytochalasin, results in rapid exocytosis after centrifugation. It is concluded that the maintenance of the spatial relationships of this organelle is mediated through the peripheral cytoskeleton.

Animals↗
PubMed1993

Rapid and accurate microfractionation of the contents of small centrifuge tubes: application in the measurement of molecular weight of proteins via sedimentation equilibrium.

The contents of small centrifuge tubes containing solutions of radiolabeled proteins that had been centrifuged to sedimentation equilibrium were fractionated using a new device based upon the mechanical design of Attri and Minton (Anal. Biochem. 152, 319-328, 1986). Individual fractions, corresponding to laminae of 0.15 mm column height within the tube, were collected using one of two methods: (a) automatic mixing with scintillation fluid and delivery to vials mounted in a fraction collector, or (b) collection of undiluted fractions on scintillation vial caps impregnated with solid scintillator. Gradients of protein concentration were obtained via scintillation counting of sequential fractions. Molecular weights of proteins ranging from 4 x 10(4) to 3.5 x 10(5) were calculated by fitting the theoretical expression for sedimentation equilibrium of an ideal homogeneous solute to the experimental gradients. The values so obtained agree well with values obtained by optical scanning of the unfractionated centrifuge tube, and with values obtained from the literature.

Animals↗
PubMed2001

Stimulation of DNA polymerase alpha by hypergravity generated by centrifugal acceleration.

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase alpha, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase alpha in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase epsilon, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase alpha reactions revealed that, under high gravity conditions, the K(m) value for template DNA decreased while the V(max) stayed constant. In contrast, the centrifugal acceleration did not affect the K(m) values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase alpha by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase alpha under low ionic conditions.

Animals↗
PubMed1978

Effect of chronic centrifugation on bone density of the dog.

Sixteen male Beagle dogs, 293 to 509 days old, were exposed almost continuously for 3 months to 2.0 G on a 7.9m radius centrifuge. The dogs were maintained on the centrifuge by means of a specially designed, automated waste disposal and life support system. Bone density was measured by a 125I Profile Scanner in the anterior, medial posterior and lateral cortex of the femoral mid-shaft. As compared to mean density in the femora of normal gravity controls, centrifuged dogs showed a 0.8% (P less than 0.05) lower mean linear absorption in their femora. However, the regression of density on the square-root of cross-sectional area/pi differs very significantly in the animals living at earth gravity and those living at hypergravity. Thin hypergravic bones are denser, thick hypergravic ones are less dense than the corresponding ones of normal gravity controls.

Animals↗
PubMed1983

Comparison of the filtration and centrifugation methods for assaying [3H](D-ala2, D-leu5)enkephalin binding to mouse brain membranes.

The binding of [3H](D-ala2, D-leu5)enkephalin (DADLE) to mouse brain membranes was studied via rapid filtration with GF/C filters and via the centrifugation method. The amount of specific binding determined by filtration was found to be dependent on the length of time between the initiation of vacuum and the actual time of filtration. Vacuum strength alone also significantly affected the amount of specifically bound [3H]DADLE measured by filtration. The centrifugation method increased the number of experimentally determined binding sites when compared to the filtration method. The difference in the number of binding sites was only partially attributable to passage of binding material through the GF/C filter. The ability of morphine to displace [3H]DADLE was also found to differ slightly between the two methods. These data indicate that centrifugation is a better method of determining the binding parameters of [3H]DADLE in mouse brain membrane preparations.

Animals↗
PubMedDate not supplied

Centrifugation of 50 ml of peritoneal fluid is sufficient for microbiological examination in continuous ambulatory peritoneal dialysis (CAPD) patients with peritonitis.

Concentration of peritoneal fluid of continuous ambulatory peritoneal dialysis (CAPD) patients has often been recommended for microbiological examination. This study presents data about the yield of microorganisms of different volumes through centrifugation. Twenty-eight samples of 50 ml and 1000 ml of the same dialysate bags were centrifuged, and assays were made for aerobic and anaerobic organisms on solid media and in enrichment broth. All pairs of 50 ml and 1000 ml samples except one (96%) showed identical results, suggesting that it is unnecessary to centrifuge more than 50 ml of fluid. Furthermore, it seems better to examine two or more consecutive bags per peritonitis episode, since microorganisms were found in seven of nine (77%) such episodes, compared to only five of eight (62%) episodes with only one bag. In addition, significantly more positive cultures were recorded (14 of 17 vs. 5 of 11; p less than 0.05) if samples were taken before administration of antibiotics. One patient's catheter was colonized with bacteria probably arranged in a biofilm. This fact seems to be of particular importance for CAPD patients and should be studied further.

Bacteria↗
PubMed1985

Estrogen receptor separation employing sucrose deuterium oxide gradients generated during sample centrifugation.

A quick and easy method of sucrose density gradient analysis is described in which prior preparation of gradients for protein separation is not required. We compare the refractive indices as well as the separation pattern of radioactively labeled proteins with either preformed gradients (prepared by layering or by gradient marker) or gradients that establish themselves during centrifugation. Our results show that an identical pattern of protein separation can be achieved by layering the nuclear extracted estrogen receptor either on top of one single sucrose concentration or on a preformed gradient. Centrifugation times are 40 hr in a swinging bucket type rotor (SW60) or 15 hr in a vertical tube rotor (VTi 65). This method avoids the onerous task of gradient preparation, and the separation itself can be achieved in a 15 hr centrifugation by use of a vertical tube rotor.

Breast Neoplasms↗
PubMed1981

Temperature regulation during centrifugal elutriation and its effect on cell separation.

Centrifugal elutriation appears to be a promising method for cell separation. The quality of the separation may be limited by the control of temperature within the separation chamber, which affects the fluid viscosity and rotor speed. The factors affecting the temperature regulations have been re-examined. At flow rates between 10 and 40 mL/min the temperature within the chamber was primarily dependent on the temperature of the fluid flowing into the rotor. Increases in the temperature of the fluid while it flowed through the rotor were observed and were greater at higher rotor speeds and lower flow rates. This heating, caused by friction at the rotating seal, could raise the fluid temperature within the chamber by as much as 6 degrees C. Fluctuations in the temperature of the centrifuge produced temperature variations of only 0.3 degrees C in the fluid in the elutriation chamber. Small increases in the rate of elutriation of cells, concomitant with centrifuge cooling and speed fluctuations, were detected by optical density measurements. However, neither the modal volume nor coefficient of variation of the collected cells were affected.

Animals↗
PubMed1999

Effects of centrifuging at 2g on rat long bone metaphyses.

Hypergravity may be considered as a means of counteracting the deleterious effects of microgravity on bone tissue. The effects of exposure to 4 days of hypergravity provided by centrifuging, on bone tissue were studied using histomorphometry. Young 53-day-old male Sprague Dawley rats were randomly divided into a centrifuged group (2g, n = 10), a rotated group (ROTATE, n = 6) of rats exposed to 1.03 g placed in cages near the centre of rotation of the centrifuge and a stationary control group (CONTROL, n = 10). The body mass of the 2g rats was decreased by this experience by 16% compared to CONTROL. The width of the tibial growth plate of 2g was decreased. In two out of ten 2g rats, the hypertrophic zone was injured. In both the tibial and humeral primary (1 degrees ) spongiosae, a reduced 1 degrees spongiosa width (-35% and -24%, ROTATE versus CONTROL respectively; -37% and -41%, 2g versus CONTROL respectively) associated with bone gain (+27% for tibia and humerus ROTATE versus CONTROL; + 16% and +20%, 2g versus CONTROL respectively) was observed in both ROTATE and 2g. In the tibial secondary (2 degrees) spongiosa, bone mass was increased in the 2g (+13% 2g versus CONTROL) rats due to thicker trabeculae, but was decreased in ROTATE rats (-12% versus CONTROL) due to thinner trabeculae. The parameters of formation and resorption activities were stimulated in the 2g and ROTATE groups, the formation activity being more enhanced in 2g. No structural changes were observed in the humeral 2 degrees spongiosa in any of the groups. Numeral bone formation parameters were decreased in 2g and ROTATE but resorption activity was increased in 2g and decreased in ROTATE compared to CONTROL. In conclusion, as early as the 4th day, 2g hypergravity induced reduced endochondral bone formation and increased cancellous bone mass. Rotation led to mixed results including reduced endochondral bone formation, increased bone volume in the 1 degrees spongiosa and bone loss in the 2 degrees spongiosa.

Animals↗
PubMed2000

Can daily centrifugation prevent the haematocrit increase elicited by 6-degree, head-down tilt?

A measure to counteract the effects of low or zero gravity is required for long-term space flight, such as the manned Mars mission scheduled by the National Aeronautics and Space administration (NASA) for 2014. We conducted a series of centrifugation experiments with humans, using a short-arm centrifuge (radius 1.8 m, made by First Medical Co., Tokyo, Japan). We employed 6-degree, head-down tilt (HDT) for 4 days to simulate space flight. Ten healthy male volunteers underwent 4-day HDT and a 2-G daily centrifuge load for 60 min in the +Gz direction and measurements, such as haematocrit, 24-h urine volume, body weight and electrocardiogram (ECG) were made. There was no significant increase in the haematocrit during the HDT period, although our previous studies had shown a significant increase during HDT. A 60-min daily load of +2 Gz appears to be effective in reversing the haematocrit increase due to 4-day HDT.

Adult↗
PubMed2005

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids.

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

Centrifugation↗
PubMed1979

The role of centrifugation in the measurement of surfactant in amniotic fluid.

Analysis of amniotic fluid fractions obtained by differential centrifugation has revealed the presence of intact globular-like structures of approximately 1 mu dimensions. These particles, which appear to be representative of fetal lung surfactant, begin precipitating at very low g forces and the extent of their removal by centrifugation was found to significantly alter L/S ratio measurements. This effect is particularly significant when moderate levels of these structures are present, which is usually between 34 and 37 weeks' gestation. False-negative L/S ratios could be attributed to this effect. Centrifugation can be employed to harvest these structures in reasonably pure form and measurement of their concentration, by phospholipid analysis, showed good correlation with fetal lung function in a trial study with 91 patients. These findings suggest that measurement of these surfactant particles harvested from amniotic fluid may provide a simple yet specific index of fetal pulmonary maturity.

Amniotic Fluid↗
PubMed1987

Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan.

A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, ind 20% Stractan; lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.

Animals↗
PubMed1989

Hemolysis by aliphatic alcohols and saponin measured by the coil planet centrifugation method.

Using the coil planet centrifugation method, the mechanism of hemolysis by alcohols and saponin was investigated. With this technique, erythrocytes are introduced into a gradient of hemolytic agents in saline, which is prepared in a long coiled polyethylene tube. The tube is centrifuged so that the cells move from a low to a high concentration of hemolytic agent. When the cells lyse, they release hemoglobin which remains stationary, and therefore hemolytic potency can be determined spectrophotometrically by the distance the cells move before lysing. We found that alcohols caused hemolysis at a particular concentration, whereas saponin-induced hemolysis was dependent on the amount of saponin accumulated in the environment of the cell. In addition, alcohols with longer carbon chains were more potent hemolytic agents than those with shorter chains, but each additional carbon group produced less of an increase in hemolysis per mole of alcohol. This chain-length dependency is consistent with a previous study on in vivo alcohol-induced hemolysis. The coil planet centrifugation method is also adaptable to comparative studies on the mechanism of other types of hemolysis, such as immune or drug-induced lysis, and to toxicological studies.

Alcohols↗
PubMed1986

Metabolic and ultrastructural characterization of guinea pig alveolar type II cells isolated by centrifugal elutriation.

Direct biochemical studies of the whole lung have been quite misleading because of the heterogeneity of the lung cell types. One of the advantages of studying the isolated cells is to be able to correlate specific metabolic functions with intracellular molecular events and to differentiate factors that affect the type II cell function directly. In the present study we have isolated type II cells from guinea pig lung with elastase and purified them by centrifugal elutriation. These cells fluoresce with phosphine 3R as the dye is specifically taken up by the lamellar bodies. In the electron micrographs, the type II cells display punctate villi, which underwent fragmentation in those cases where metrizamide density gradient was used. Mitochondria are scattered throughout the cytoplasm, and smooth endoplasmic reticulum is sparse. Type II cells possess large irregularly shaped nuclei with peripheral areas of dense hemochromatin and at least one prominent nucleolus. Ovoid lamellar bodies are the most prominent cellular inclusions. These bodies are present throughout the cytoplasm and contain a substructure of whorling and concentric laminations. Biochemical studies indicate that type II cells prepared by centrifugal elutriation are metabolically well preserved as seen from incorporation of [14C]leucine into cellular proteins, [methyl-14C]choline into cellular disaturated phosphatidylcholine and CDP[methyl-14C]choline into mitochondrial and microsomal phosphatidylcholine. Superiority of centrifugal elutriation over the commonly employed combination of discontinuous metrizamide gradient and cell elutriation is evident from the present study.

Animals↗
PubMed1979

Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations.

Both normal and leukemic human lymphoid cell lines were separated into populations corresponding to different positions in the cell cycle by centrifugal elutriation. Each population was analyzed for cell concentration, cell volume, [3H]thymidine incorporation, percentage S phase by autoradiography, and percent G1, S, and G2/M phases by flow cytometry. The smallest cells, collected at the lowest flow rate, were in G1 phase. Cells collected at increasing flow rates progressively increased in volume and represented distinct positions in the cell cycle transition from G1 phase, through S phase, and into G2/M phase. The purity of the G1 population varied according to cell load. One hundred percent of cells were recovered and cells collected in G1- and S-phase populations proliferated in culture with patterns characteristic of synchronized cells. An aneuploidy leukemia cell line, CEM, was separated into near-diploid and near-tetraploid populations by centrifugal elutriation. This method of cell separation provides large numbers of human lymphoid cells at different positions in the cell cycle for investigating the relationship between the cell cycle and various surface membrane and metabolic properties of cells. Aneuploid leukemia and lymphoma cells can be separated by centrifugal elutriation into populations which contain different numbers of chromosomes for comparisons of their biologic properties.

Aneuploidy↗