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At least 1,189 records · Page 66Linked to original sources
PubMed1982

Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll density gradient centrifugation.

Techniques were developed for the isolation of human neutrophils from large volumes of human blood. This preparative procedure avoids exposure to Ficoll/Hypaque and offers a low cost alternative to centrifugal elutriation. First, leukocyte-enriched suspensions were obtained by treating buffy coats with 2% dextran. The leukocyte suspensions were then fractionated on three steps Percoll density gradients. The gradients were designed to carry large numbers of cells while maintaining resolution of the cell peaks. As a result, at least 320 X 10(6) leukocytes could be processed on a single 12 ml gradient. Extensive testing of the procedure showed that the purity of the neutrophil band was 96.5 +/- 3.16% (n = 50). The band contained 82.4 +/- 14.6% (n = 39) of the neutrophils applied to the gradient. Viability assays (bacterial killing, chemotaxis) demonstrated that neutrophils isolated on Percoll gradients were just as active as those harvested by centrifugal elutriation. Recovery data are presented and compared to the results obtained by others.

Blood Bactericidal Activity↗
PubMed1984

Evaluation of the EMIT reagent system for measurement of caffeine with the EMIT Lab 5000 System and a centrifugal analyzer.

Using a Syva EMIT Lab 5000 System, we evaluated a newly developed EMIT assay for measurement of caffeine in serum of neonates. The method is rapid, and coefficients of variation for within-run and day-to-day precision were less than 5%. The accuracy of the method, as evaluated from analytical recovery and a calibrator-crossover study, is excellent. Comparison of the results analyzed by the EMIT caffeine assay (y) and the liquid-chromatographic comparison method (x) yielded the linear regression equation y = 1. 052x + 0.293 (n = 110, r = 0.987, and Sy . x = 0.625). The method was also adapted to a COBAS-BIO centrifugal analyzer (y) and optimized to minimize sample and reagent consumption. Linear regression analysis yielded the equation y = 1. 031x + 0.227 (n = 61, r = 0.992, and Sy . x = 0.488). As many as 400 assays per EMIT kit can be performed with the analyzer method, with excellent precision and accuracy. The kit assay and this centrifugal analyzer are well suited for rapid batch testing: 28 analyses (including six calibrators) in 10 min.

Adult↗
PubMed1983

Characterization of rat lymphocyte cell membranes by analytical isopycnic centrifugation.

Lymph node cell homogenates were fractionated by differential or isopycnic centrifugation and the fractions analyzed for biochemical markers with particular focus on plasma membrane constituents. Markers for the nucleus (DNA), mitochondria (cytochrome oxidase), and lysosomes (acid hydrolases) showed the expected distributions which were different from those of membrane-bound enzymes. 5'-Nucleotidase, alkaline phosphodiesterase, gamma-glutamyltranspeptidase, and cholesterol were membrane-bound and distributed identically after isopycnic centrifugation with peaks at 1.15. The distributions of the enzymes were all shifted to higher densities by digitonin treatment, confirming their association with plasma membrane-derived elements. The distribution of galactosyltransferase (ovalbumin acceptor), largely overlapped those of plasma membrane markers but it was only slightly shifted by digitonin, suggesting its localization in Golgi apparatus. The distribution of mannosyltransferase (dolichyl phosphate acceptor) also overlapped those of plasma membrane and Golgi markers but it was centered at higher density (1.18) and was unaffected by digitonin. It is a useful marker for endoplasmic reticulum. 50% of the activity was in low speed "nuclear" sediments where it was associated with the nuclear membrane. A number of other putative and previously used markers for the endoplasmic reticulum of lymphocytes were shown not to be localized in these membranes. In particular, NADH-cytochrome c reductase was only partly associated with the endoplasmic reticulum (56%) and the remainder of the activity was in mitochondria (44%). The results show the heterogeneity in equilibrium density of plasma membrane vesicles and the considerable overlap of their distribution with those of other cellular membranes; they should provide a basis for the more rational design of preparative procedures for the lymphocyte plasma membrane.

Animals↗
PubMed1984

Automated prothrombin-time test with use of a chromogenic peptide substrate and a centrifugal analyzer.

We modified a test for prothrombin time, to automate its performance by centrifugal analysis. The sample is activated with calcium-thromboplastin in the presence of a thrombin-specific chromogenic peptide substrate, Tos-Gly-Pro-Arg-p-nitroaniline (Chromozym TH). The endpoint of the reaction is set as the time after onset until a defined amount of substrate has been cleaved, as reflected in a fixed change in absorbance. The assay is as sensitive as clotting-type methods are to the clotting factors of the extrinsic pathway--except for fibrinogen, which can be estimated from the maximum absorbance difference. Substrate concentration has little effect on the result, and varying it does not significantly change the sensitivity to factor X, which indicates little interference with the enzymes of the coagulation cascade. The method was applied to the Cobas Bio centrifugal analyzer. An external calculator must be used to convert absorbance readings into reaction times, which can be expressed as percent-of-normal values or as "ratios" by use of reagent-specific factors. CVs were less than 2% within run, less than 6% day to day. Overall correlation with the clotting test (r = 0.95) was good. Using the Cobas Bio, we achieved a throughput rate of 200 samples per hour.

Autoanalysis↗
PubMed1980

Coil planet centrifuge - a new method for measurement of osmotic fragility of erythrocytes and its application in alcohol liver injuries.

The present study was designed to examine the osmotic resistance of erythrocytes measured by a new method-Coil Planet Centrifuge under the glutaraldehyde incubation, and to determine free cholesterol and adenine nucleotide contents of glutaraldehyde-treated erythrocytes compared with that of control erythrocytes. Glutaraldehyde increased the osmotic resistance of erythrocytes. Hemolysis end point of glutaraldehyde-treated erythrocytes shifted to hypo-osmolar side after 2.5 h hours incubation (p less than 0.001). Free cholesterol content of erythrocytes was not changed by the addition of glutaraldehyde, however, ATP content significantly increased when compared with controls (p less than 0.001). Moreover, the osmotic resistance of erythrocytes in 55 patients with alcoholic liver injuries and the relation between the disease were examined by Coil Planet Centrifuge. Hemolysis end point shifted to hypo-osmolar side in the patients, and our results suggest that the increase in osmotic resistance is partly responsible for the increase in the rigidity of erythrocytes. This new method was also helpful in evaluation in the clinical course of alcoholic liver injuries.

Adenosine Triphosphate↗
PubMed1981

Platelet monoamine oxidase. I: Effect of temperature, anticoagulant, and centrifugation technique.

Platelet yield and monoamine oxidase (MAO) activity per platelet in platelets obtained by the method of Corash, which isolates essentially all platelets from whole blood, and by a single differential centrifugation of whole blood, were compared. Platelets recovered by one spin in a swinging bucket rotor appeared to be a homogeneous population containing a reliable representation of the platelet population obtained by the Corash method since the MAO specific activity yielded by the two methods was not significantly different. The effect of the anticoagulant, temperature, and type of rotor on platelet recovery from blood and MAO activity/platelet were also studied. The MAO and plasma amine oxidaze (PAO) activities obtained from blood anticoagulated with ethylenediaminetetracetate (EDTA) were significantly greater than those found when acid-citrate-dextrose was the anticoagulant. Blood centrifuged in a swinging bucket rotor at room temperature yielded 80-90% of total platelets as compared to the 45-65% recovered with a fixed-angle rotor at 0-4 C. The most efficient method of platelet isolation was found to be a 2 1/2-min room-temperature spin at 600 X g with a swinging bucket rotor using EDTA as anticoagulant.

Adult↗
PubMed1982

Determination of the specific gravity of certain helminth eggs using sucrose density gradient centrifugation.

The specific gravities of ten species of helminth eggs were determined using sucrose density gradient centrifugation. Fecal or egg concentrate was layered over a 3 to 54% sucrose density gradient. The gradient was then centrifuged at 800 g for 20 min, allowing 5 min for acceleration and 5 for deceleration. Bands formed were identified and measured. Refractive index was measured at the middle of narrow bands, or at the level at which the concentration of eggs was highest, in the case of wide bands or when no band was formed. The specific gravity corresponding to this refractive index was taken as the specific gravity of the eggs. The ten species of helminth eggs studied and specific gravities measured on three or four gradients were: Toxascaris leonina, 1.0559; Ancylostoma caninum, 1.0559; Toxocara canis, 1.0900; Parascaris equorum, 1.0969; Toxocara cati (embryonated), 1.1005; Ascaris suum, 1.1299; Trichuris suis, 1.1299; Trichuris vulpis, 1.1453; Taenia sp., 1.2251; and Physaloptera sp., 1.2376. These determinations agree with or approximate those of previous workers. The specific gravities of P. equorum, T. suis, Taenia sp., and Physaloptera sp., are reported for the first time.

Animals↗
PubMed1981

Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.

The first commercially available centrifugal analyzer having fluorescence detection capability was used to develop kinetic fluorometric assays for several proteinases. The substrates were all synthetic oligopeptides incorporating 7-amino-4-trifluoromethylcoumarin, a molecule that can be detected either spectrophotometrically or fluorometrically. We thus compared the centrifugal analyzer spectrophotometric and fluorometric detection systems, finding fluorescence detection to be 50-fold more sensitive. We also compared the sensitivity of the fluorescence detector to that of a conventional spectrofluorometer by determining the minimum detection limit for each enzyme on both instruments; we found them to be similar in sensitivity. As an illustrative application, we measured the cathepsin B-like activity in serum samples from 55 women. The median enzyme activity of women taking oral contraceptives and pregnant women was increased two- and threefold, respectively, over the control group (about 5% CV within run, and 10% CV between runs).

Autoanalysis↗
PubMed1982

Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation.

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.

Animals↗
PubMed1982

Enrichment by counterpart centrifugal elutriation of human lymphocytes cytotoxic to human tumour cells.

The enriched fractions of cytotoxic cells responsible for natural killer (NK) activity against both human sarcoma and neuroblastoma (LA-N2) cell lines were readily obtained by countercurrent centrifugal elutriation (CCE). The NK cells were obtained in the larger lymphocyte fractions (fraction 6 +/- 1), having a mean cell volume of 180 u3. The cytotoxic-enriched fraction contained 51% large lymphocytes having cytoplasmic granules. On the other hand, monocytes were purified to greater than 90% and isolated in another fraction (final fraction) and these cells had the lowest NK activity against both human tumour cell lines. However, compared with the lymphocyte fractions, small and large monocytes displayed greater antibody-dependent cellular cytotoxicity (ADCC) activity against human B erythrocytes. These results indicate that NK found to have activity against both tumour cells lines were larger lymphocytes, not small monocytes. Thus, countercurrent centrifugal elutriation (CCE) can provide a sensitive method to obtain enriched fractions of large lymphocytes contained tumoricidal activity against human sarcoma and neuroblastoma cell lines.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1978

Manual and centrifugal analyzer determination of cholesterol and triglycerides with single-vial enzymatic reagents.

We have assessed Calbiochem single-vial enzymatic reagent systems for the analysis for cholesterol and triglycerides, using manual and centrifugal analyzer techniques. Analysis for cholesterol by both techniques had acceptable performance by the short-term criteria of the Center for Disease Control (CDC) during this study carried out under optimal conditions of variance. Analysis for triglycerides by the manual technique did not meet CDC criteria, and we do not recommend use of this method. Analysis for triglycerides by the centrifugal analyzer technique had acceptable short-term precision but had unacceptable accuracy at high concentrations of triglycerides; this method may be considered to have acceptable performance for routine laboratory use.

Centrifugation↗
PubMed1982

Isolation of ovine lymphocytes, granulocytes, and monocytes by counterflow centrifugation elutriation.

A simple, brief 1-step counterflow centrifugation elutriation technique is described for the recovery of highly purified populations of lymphocytes and granulocytes from adult and fetal ovine peripheral blood. Monocytes are obtained as an enriched population. Lymphocyte and granulocyte recoveries average 80%, higher than with routine gradient centrifugation procedures. In this described technique, inexpensive medium is used, and it is completed in about 90 minutes. The individual blood cellular populations, when tested appropriately, demonstrated normal functional responses.

Animals↗
PubMed1977

Separation of leukemic cells into proliferative and quiescent subpopulations by centrifugal elutriation.

Centrifugal elutriation was used to separate human acute leukemia cells into proliferative and quiescent subpopulations. Ten bone marrow specimens and 5 peripheral blood specimens were subjected to centrifugal elutriations. From each patient, leukemic cell subpopulations were obtained for which the [3H]thymidine labeling index differed by 10- to 30-fold. In 6 of the marrow specimens and in 2 of the peripheral blood specimens, cell subpopulations were obtained for which the labeling index exceeded 20%. In 5 marrow specimens, subpopulations were obtained for which the labeling index exceeded 40%. Preliminary studies of the uptake of 1-beta-D-arabinofuranosylcytosine and 5-azacytidine failed to show any correlation between drug uptake and the proliferative characteristics of the leukemic subpopulations.

Azacitidine↗
PubMed1982

Development of a multichannel electrochemical centrifugal analyzer.

A new instrument has been developed that is similar in design and concept to the GeMSAEC centrifugal analyzer (Oak Ridge National Laboratory) except that electrochemical detection is used rather than optical detection. The present version has eight channels, each with its own detector. Problems of speed control and sampling synchronization are greatly minimized from those of a multichannel centrifugal analyzer having a single detector system. All eight sample compartments and polarographic cells are contained in a single Teflon rotor, 7.7 cm in diameter. The working electrodes are planar carbon electrodes. The initial application of the analyzer is in kinetic methods involving the rates of enzyme-catalyzed reactions. The initial system chosen was the glucose oxidase system. When the rotor is spun, the solution moves into the sample cell and up against the planar working electrode. The reaction starts and current is measured as a function of time at a constant applied voltage. The current output from each polarographic circuit is connected to a separate channel of a multiplexed analog-to-digital converter. The analyzer is controlled by a dedicated microcomputer system, which sets the polarographic cell voltages, collects the current-time data, and calculates the results.

Autoanalysis↗
PubMed1982

G-tolerance enhancement: straining ability comparison of aircrewmen, nonaircrewmen, and trained centrifuge subjects.

G tolerance measurements, both relaxed and using a protective straining maneuver, were made on aircrewmen, nonaircrewmen, and trained centrifuge subjects at the USAF School of Aerospace Medicine over the past 3 years. Results indicated that currently trained pilots (including fighter-type pilots) are not as proficient at performing a G protective straining maneuver as fully trained centrifuge panel members. Pilots should be able to gain at least 2 G over relaxed G tolerance with adequate training. Evidence exists to indicate an optimum time for G training would be during the period of undergraduate pilot training.

Acceleration↗
PubMed1978

Kinetic centrifugal analyzer and manual determination of serum urea nitrogen, with use of o-phthaldialdehyde reagent.

o-Phthaldialdehdye reagent was used in assaying urea with a miniature centrifugal analyzer. Seventeen samples can be assayed kinetically in 2 min at a current cost of less than a penny per test. The reaction is initiated in the instrument upon simultaneous mixing of the o-phthaldialdehyde reagent containing 10 microliter of sample with dilute sulfuric acid/N-(1-naphthyl)ethylenediamine dihydrochloride reagent. The reaction is monitored kinetically at 546 nm during the initial 2 min, when the rate is nearly constant. The reaction of o-phthaldialdehyde with urea reaches equilibrium by about 7 or 8 min, but the method cannot be used for end-point analysis because of interference by the blank reaction. A 1.2 g/liter solution of o-phthaldialdehyde used in conjunction with 2 mol/liter sulfuric acid solution was found to be most suitable for centrifugal analyzers. I also describe a manual version of the procedure that gives a linear standard curve up to 1.0 g of urea nitrogen per liter.

Centrifugation↗
PubMed1981

[Development of a gentamicin producer under different culture conditions studied by a method of differential centrifugation of the mycelium in a saccharose density gradient].

The method of differential centrifugation in the sucrose density gradient (SDG) enabled one to trace the changes in the development of the seed and fermentation mycelium of the gentamicin-producing organism. Correlation between gentamicin distribution in the SDG and the culture productivity was found. It was shown that the culture grown under the optimal aeration and agitation conditions was characterized by formation of higher amounts of the mycelium in the 5th and 6th layers of the SDG. Such mycelium was more productive than that from the other SDG layers. The most productive 48-hour seed culture had the more significant part of the mycelium in the 3rd layer of the SDG. When such a culture had the more significant part of the mycelium in the 3rd layer of the SDG. When such a culture was used as the seed material, the activity of the fermentation broth was the highest. The method of differential centrifugation in the SDG provides determination of the culture productivity by the volumes of the fermentation mycelium in the 5th and 6th layers or the seed mycelium in the 3rd layer of the SDG.

Centrifugation, Density Gradient↗
PubMed1981

Effect of chronic centrifugation on the cross-sectional shape of long bones in dogs.

Alterations in cross-sectional shape at midlength of the femur, tibia, and humerus of 16 male Beagle dogs exposed for 6 months to either 2.1 g or 2.6 g by centrifugation were analysed by the area moment correlation coefficient, as defined by AMTMANN and POTULSKI (1981). As shown by variance analysis the cross-sectional shape differences between bones are highly significant, while the treatment effects are only significant at a 5% level. While the femur and the tibia are almost alike in the shape of their whole sectional area and cortical surface, they differ in the shape of their marrow cavity. The most oval cross sections were found in the humerus. In femur and humerus the shape of the whole sectional area, the marrow cavity and the cortical surface tend to be more oval in the centrifuged animals than in the controls. In the tibial cross sections the opposite is the case. A significant interaction "Gravity X Bone" was found for the shape of the marrow cavity.

Analysis of Variance↗