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PubMed1970

Centrifugal sedimentation of virus particles for electron microscopic counting.

Centrifuge cells with conical chambers were provided by using special inserts for the stainless-steel tubes that fit the Spinco SW-39 rotor. Particulate material, centrifuged in these cells, was collected on carbon-coated glass discs. These discs were exposed to OsO(4) vapor, dehydrated in graded alcohols, air-dried, and metal-shadowed. The metal-shadowed carbon film was floated from the glass, mounted on a grid, and examined. A knowledge of cell geometry and microscope magnification allowed correlation of the number of particles observed to a volume of the original suspension. A precision of +/-6% at the 95% confidence level was attained when counting approximately 100 particles per 10,000 x field. Applications and advantages of the method are discussed.

Centrifugation↗
PubMed1968

Platelet adhesiveness: the effect of centrifugation on the measurement of adhesiveness in platelet-rich plasma.

Platelet adhesiveness has been measured in citrated whole blood and in platelet-rich plasma obtained from normal subjects, splenectomized patients, and from patients in whom the diagnosis of recurrent venous thrombosis had been made. The duration of centrifugation used in the preparation of platelet-rich plasma was found to have a profound effect on the measurement of platelet adhesiveness because the figure for platelet adhesiveness measured in platelet-rich plasma obtained by centrifugation was considerably lower than that found in citrated whole blood. This effect was particularly marked when platelet-rich plasma was obtained from subjects in whom platelet adhesiveness measured in whole blood was increased.

Blood Cell Count↗
PubMed1974

Separation of abnormal cells in the peripheral blood by means of the zonal centrifuge.

The basic principles of rate centrifugation in the large volume zonal centrifuge and its application to the separation of abnormal or rarely occurring cells in the blood are described. Details of the method, gradient media, and collection and examination of the fractions obtained are given. A description of the cells found in normal human blood, in a subject undergoing known immunological stimulus, and in glandular fever and Hodgkin's disease is given and the implications are discussed.

Antigens↗
PubMed1998

A pseudo-cryptococcal artefact derived from leucocytes in wet India ink mounts of centrifuged cerebrospinal fluid.

Wet India ink mounts of cerebrospinal fluid (CSF) are useful in the laboratory diagnosis of cryptococcal meningitis. Pseudo-cryptococcal artefacts in such mounts have been attributed to leucocytes in CSF but their mode of formation has not been explained. This report describes the reproduction of such an artefact in cryptococcus free CSF-leucocyte mixtures that had been subjected to high speed centrifugation. The viscosity of DNA that could provide a morphological pseudo-capsule, and the yellow-green fluorescence of the pseudo-capsular material on staining with acridine-orange, suggest that lymphocytic nuclear DNA, which possibly leaked out after damage to the lymphocyte membrane by centrifugation, was responsible for this artefact.

Artifacts↗
PubMed1991

Recovery of gravitropism after basipetal centrifugation in protonemata of the moss Ceratodon purpureus.

Apical cells of 5-day-old dark-grown protonemata of the moss Ceratodon purpureus (Hedw.) Brid. are negatively gravitropic and appear to utilize amyloplasts as statoliths. These cells exhibit a characteristic plastid zonation (five zones) with one zone (No. 3) specialized for the lateral sedimentation of amyloplasts. Basipetal centrifugation displaces all amyloplasts in the apical cell to the end wall. In basipetally centrifuged protonemata observed using infrared videomicroscopy, tip extension occurred with or without amyloplasts present in the apical dome. The initial return of upward curvature was always correlated with the return and sedimentation of amyloplasts in zone 3. Subsequent vigorous upward curvature was correlated with distinct amyloplast zonation and further sedimentation in zone 3. Initial downward ("wrong way") curvature, which often preceded upward curvature, correlated with the presence of amyloplasts in the apical dome (zone 1). These data support the hypotheses that nonsedimenting amyloplasts in zone 1 are necessary for initial downward curvature and that amyloplast sedimentation in zone 3 is necessary for upward curvature.

Centrifugation↗
PubMed2002

Endotoxin-free purification of monocytes for dendritic cell generation via discontinuous density gradient centrifugation based on diluted Ficoll-Paque Plus.

BACKGROUND: For the investigation of the biology of human monocyte-derived dendritic cells (DCs), and also for further clinical use of these cells, it is important to separate large numbers of monocytes without isolation-induced activation. METHODS: Monocytes were purified from mononuclear cells (MNCs) by density gradient centrifugation using Ficoll-Paque Plus adjusted to a density of 1,068 g/ml by dilution with phosphate-buffered saline (PBS) and compared to Percoll isolated monocytes. DCs were generated by culturing monocytes with interleukin (IL)-4 plus granulocyte-macrophage colony-stimulating factor (GM-CSF), and they were functionally tested for IL-12 p70 production by stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). RESULTS: Density gradient centrifugation using diluted Ficoll-Paque Plus resulted in a monocyte purity of more than 75% and a recovery of 70%. DCs derived from such monocytes produced high amounts of IL-12 p70 in response to LPS plus IFN-gamma, thus demonstrating an important functional capacity. CONCLUSION: Since Ficoll-Paque Plus is commercially available in an endotoxin-free form, this modified Ficoll gradient offers a simple modality for isolating large amounts of monocytes under endotoxin-free conditions without introducing further potential monocyte-activating agents or procedures.

Cell Differentiation↗
PubMed1980

Isopycnic centrifugation of human, pigeon and guinea pig erythrocytes.

The buoyant densities of human erythrocytes with normal (HbAA) and abnormal heterozygous haemoglobins (HbSS, HbAS, HbSC) as well as erythrocytes from guinea pig and pigeon were determined by centrifugation in discontinuous density gradients of metrizamide. Guinea pig, pigeon and human erythrocytes from normal human adult (HbAA) and from heterozygote HbAS were found to have the same density of 1.1381 g/cm3, while erythrocytes from a sickle-cell patient (HbS) and from a heterozygote HbSC, HbSS and HbSC, which are known to have a higher lipid content than isovolumic normal cells, had a lower density of 1.1261 g/cm3. When centrifugation was carried out under non-equilibrium conditions (rate zonal), several sedimentation bands were obtained from each erythrocyte preparation. Differences in the sedimentation rates were unrelated to the haemoglobin composition of the erythrocytes. It is suggested that the lower density of the erythrocytes associated with sickle-cell disease may have significant consequences in the circulation of such cells and in the clinical sequence of the sickling process.

Anemia, Sickle Cell↗
PubMed1992

Superiority of gel centrifugation in antibody screening and identification.

We report on the direct comparison of gel centrifugation technique and tube testing for antibody screening (ABS) under controlled routine conditions. 3,000 blood samples were screened for antibodies (AB) by gel centrifugation (ID-System, bromelin 37 degrees C and room temperature, indirect antiglobulin test with LISS) and a sensitive tube test (TT; bromelin two-phase test, 37 degrees C and room temperature, and indirect antiglobulin test with 22% bovine albumin) in parallel. By ID significantly more relevant and potentially hemolytic AB (51 vs. 35 AB/1.7 vs. 1.2%) could be detected: anti-E 4, -C 1, -D 4, -CW 2, -c 2, -Jk(a) 2, -Jk(b) 1. Eleven of these even remained negative in TT when retested with increased sensitivity and taking additional (homozygous) test cells. In addition, naturally occurring but rarely hemolytic AB (35 vs. 23 AB/1.2 vs. 0.8%) were more frequently detectable by ID: anti-Le(a) 6, -Le(b) 2, -P1 6. In contrast, only two AB were only positive in TT: anti-Le(a) 1, -Le(a, b) 1. The main disadvantage of the ID was its frequent positivity (7.7 vs. 4.3%) due to irrelevant cold AB (anti-I, -HI, -H) and unspecific factors. This can be partly reduced by omission of the bromelin test at room temperature (ID 3.0%, TT 1.5%) as the detection of relevant AB is not affected. The frequency of naturally occurring AB was still the same as in TT (0.7%) when bromelin at room temperature was omitted in both techniques. Further advantages of the ID are simplicity, small volumes of sera and reagents, and easy evaluation.

Blood Grouping and Crossmatching↗
PubMed1996

Purification of mast cells with an improved nonsynchronous flow-through coil planet centrifuge.

A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.

Animals↗
PubMed1990

Separation of bovine bronchial epithelial cell subpopulations by density centrifugation: a method to isolate ciliated and nonciliated cell fractions.

Bronchial epithelial cells isolated by protease digestion can be cultured in vitro for the study of proliferation and differentiation. However, these cells represent a heterogenous population, the components of which likely interact with one another. We attempted to utilize density gradient centrifugation as a method to prepare subpopulations of these bronchial epithelial cells. The suspension of the cells obtained by protease digestion of the bovine bronchi was mixed with an equal volume of colloidal silica reagent, Sepracell-MN, and centrifuged to form a continuous density gradient. Two distinct cell layers were identified in addition to a cell pellet at the bottom. Cells from fraction A (top layer) were more than 95% ciliated cells by morphologic examination. These ciliated cells were recovered intact as assessed by trypan blue dye exclusion and by watching beating of their cilia. The cells from fraction C (bottom layer) were 89.9 +/- 3.88% nonciliated, small round cells with a densely staining nucleus and scant cytoplasm. Comparison of cell morphology of these cells with basal cells in vivo and electron microscopic examinations suggested that these cells were basal cells. These basal cells showed an exponential cell proliferation until confluence in Ham's F12 with supplements, LHC9, and a 1:1 mixture of Medium-199 and modified Eagle's medium with 2% fetal calf serum. In contrast, the cells from fraction A grew minimally in all conditions tested. This difference was also shown in the study of DNA synthesis by [3H]thymidine uptake. Enzyme-linked immunosorbent assay for release of bovine fibronectin into cultured media indicated that fraction C cells secreted much more fibronectin (532 +/- 5.28 ng/10(6) cells/h) than fraction A cells (73.4 +/- 1.00). We also used Percoll as a density-gradient reagent and showed potential usefulness in the preparation of cell fractions of bronchial epithelial cells. In conclusion, it was possible to separate ciliated and nonciliated, presumably basal, cells of bovine bronchial epithelial cells. These differed in growth and fibronectin secretion. Studies of airway cell biology may be aided by the availability of more homogenous cell populations.

Animals↗
PubMed1991

Comparison of the standard roller pump and a pulsatile centrifugal pump for extracorporeal circulation during routine coronary artery bypass grafting.

The present prospective study compared the standard nonpulsatile twin roller pump with the Sarns centrifugal pump, in the pulsatile mode, as arterial pumps for extracorporeal circulation during coronary artery bypass grafting (CABG). The study was conducted in two consecutive groups of 25 patients receiving a standard anaesthetic and surgical protocol. The investigated parameters included haemodynamic profiles, oxygen exchange, blood gas and acid-base homeostasis, haematology, coagulation and complement consumption. With comparable settings for pump flow, gas flow and delivered oxygen concentrations, there was no difference between the groups in the main haemodynamic parameters during cardiopulmonary bypass (CPB). However, a tenfold lower dose of sodium nitroprusside was required to keep systemic vascular resistance within physiologic limits during CPB in the centrifugal group (C group) compared with the roller group (R group). During rewarming oxygen extraction was higher in the C group than in the R group. During the first eight hours after CPB no differences in haemodynamics, oxygenation parameters and pulmonary shunt between the groups were observed. During, as well as after, CPB there was no significant difference in blood gas and acid-base homeostasis between either group. Average postoperative blood loss via chest tubes, total transfusions of blood products, haemoglobin and coagulation did not differ between the two groups. However, the white blood cell count, corrected for changes in haematocrit, decreased during the early phase of CPB in the R group, but not in the C group.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1995

Comparison of the effects of centrifugal versus roller pump on the immune response in open-heart surgery.

We compared the effects of a centrifugal pump with those of a roller pump on immune responses in 26 coronary artery bypass surgery patients during cardiopulmonary bypass (CPB). The patients were randomly allocated into a (Biomedicus) centrifugal pump group and a (Stöckert) twin roller pump group. Leucocyte and differential counts; percentages of lymphocyte subpopulations (CD3-, CD4-, CD8-, CD16-, CD20- and CD25-positive lymphocytes) and monocytes (CD14); phytohaemagglutinin-, concanavalin A-, and pokeweed mitogen-induced and unstimulated proliferation of separated lymphocytes; unstimulated and pokeweed mitogen-stimulated production of IgG, IgM, or IgA; and plasma fibronectin, C-reactive protein and serum albumin concentrations were measured preoperatively, immediately before CPB, immediately before aortic declamping and on the first postoperative morning. Significant changes were seen in these variables, but no differences occurred between the groups.

Aged↗
PubMed2003

Platelet dysfunction in cardiopulmonary bypass: an experimental comparative study between a centrifugal and a new pulsatile pump.

The aim of this investigation was to study the effect of a new pulsatile pump for extracorporeal circulation (ECC) on platelet count and platelet function with respect to a Biomedicus centrifugal pump. Thirteen pigs, 8 in the pulsatile group (PG) and 5 in the centrifugal group (CG), underwent a partial extracorporeal circulation lasting 3 h. The animals were sacrificed 3 h post-ECC. The platelet study was both quantitative (platelet count) and qualitative (platelet function analysis) by assessing the closure time (CT) with a PFA-100 system. The decrease in platelet number from basal to 3 h post ECC was only significant in CG (p = 0.009). The platelet function was impaired in both groups, but the value of CT with col/ADP increased significantly only in CG (p < 0.001). The increase of CT with col/EPI was greater in CG (p = 0.07) than in PG (p = 0.2). The results indicated that the new pulsatile pump preserves platelets quantitatively and qualitatively well compared to a Biomedicus pump.

Acid-Base Equilibrium↗
PubMed2004

Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine.

BACKGROUND: Sucrose density gradient centrifugation and cross-flow filtration methods have been developed and standardised for the safe and reproducible production of inactivated arbovirus antigens which are appropriate for use in diagnostic serological applications. METHODS: To optimise the maximum titre of growth during the propagation of arboviruses, the multiplicity of infection and choice of cell line were investigated using stocks of Ross River virus and Barmah Forest virus grown in both mosquito and mammalian cell lines. To standardise and improve the efficacy of the inactivation of arboviral suspensions, stocks of Ross River virus, Barmah Forest virus, Japanese encephalitis virus, Murray Valley encephalitis virus and Alfuy virus were chemically inactivated using binary ethylenimine at a final concentration of 3 mM. Aliquots were then taken at hourly intervals and crude inactivation rates were determined for each virus using a plaque assay. To ensure complete inactivation, the same aliquots were each passaged 3 times in Aedes albopictus C6/36 cells and the presence of viral growth was detected using an immunofluorescent assay. For larger quantities of viral suspensions, centrifugation on an isopycnic sucrose density gradient or cross-flow filtration was used to produce concentrated, pure antigens or partially concentrated, semi-purified antigens respectively. RESULTS: The results of the propagation experiments suggested that the maximum viral titres obtained for both Ross River virus and Barmah Forest virus were affected by the incubation period and choice of cell line, rather than the use of different multiplicity of infection values. Results of the binary ethylenimine inactivation trial suggested that standardised periods of 5 or 8 hours would be suitable to ensure effective and complete inactivation for a number of different arboviral antigens. CONCLUSION: Two methods used to prepare inactivated arbovirus antigens have been standardised to minimise production failure and expenditure and to provide reagents that conform to the highest quality and safety requirements of a diagnostic serology laboratory. The antigens are suitable for use in either enzyme linked immunosorbent assays or haemagglutination inhibition assays and the optimised protocols can be directly applied to produce antigens from new or emerging arboviral pathogens.

Aedes↗
PubMed1984

Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation.

To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.

Animals↗
PubMed1988

Enrichment of corticotropes by counterflow centrifugation.

Anterior pituitary corticotropes represent only 9-10% of the mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells. The separation was done by counterflow centrifugation in an elutriator containing the Sanderson chamber which was designed to separate small cells under 15 micron in diameter. The corticotropes were initially eluted at flow rates under 30 ml/min along with other cells less than 12.5 micron in diameter. They were then stimulated for 2-4 h with 0.5 nM CRH and reeluted with the use of higher flow rates to separate the enlarged corticotropes from the unstimulated cells. Reelutriation of the entire pool of small cells produced an enrichment to 60% corticotropes in five separate experiments. However, when the pool was divided into smaller cells (eluted at 20 ml/min) and medium-sized cells (eluted at 30 ml/min), and the two pools were reeluted separately, the enrichment increased to over 90% corticotropes in eight separate experiments. These corticotrope populations remained enriched for up to 14 days in culture. They also secreted in a reverse hemolytic plaque assay that recognizes ACTH-(25-39). The dual labels for ACTH and beta-endorphin showed that 60% of the corticotropes stored both peptides, whereas 30% stored only ACTH, and 10% stored only beta-endorphin. No differences in storage patterns were seen when small and medium-sized corticotropes were compared. Thus, these studies present the first report of the production of an enriched fraction of CRH-responsive corticotropes by counterflow centrifugation and the first report of heterogeneous storage of ACTH and beta-endorphin. The use of enriched fractions facilitated the analysis of these heterogeneous storage patterns in over 8000 corticotropes.

Adrenocorticotropic Hormone↗
PubMed2000

Attachment forces of ants measured with a centrifuge: better 'wax-runners' have a poorer attachment to a smooth surface.

The symbiotic ant partners of glaucous Macaranga ant-plants show an exceptional capacity to run on the slippery epicuticular wax crystals covering the plant stem without any difficulty. We test the hypothesis that these specialised 'wax-runners' have a general, superior attachment capacity. We compared attachment on a smooth surface for 11 ant species with different wax-running capacities. The maximum force that could be withstood before an ant became detached was quantified using a centrifuge recorded by a high-speed video camera. This technique has the advantage of causing minimum disruption and allows measurements in very small animals. When strong centrifugal forces were applied, the ants showed a conspicuous 'freezing reflex' advantageous to attachment. Attachment forces differed strongly among the ant species investigated. This variation could not be explained by different surface area/weight ratios of smaller and larger ants. Within species, however, detachment force per body weight (F/W) scaled with the predicted value of W(-)(0.33), where W is body weight in newtons. Surprisingly, our results not only disprove the hypothesis that 'wax-runners' generally attach better but also provide evidence for the reverse effect. Superior 'wax-runners' (genera Technomyrmex and Crematogaster) did not cling better to smooth Perspex, but performed significantly worse than closely related congeners that are unable to climb up waxy stems. This suggests an inverse relationship between adaptations to run on wax and to attach to a smooth surface.

Animals↗
PubMed2001

Copper transport by lobster hepatopancreatic epithelial cells separated by centrifugal elutriation: measurements with the fluorescent dye Phen Green.

The hepatopancreas of the American lobster (Homarus americanus) possesses four types of epithelial cells arranged along blind-ended tubules. At the distal tips of these tubules, stem cells termed E-cells differentiate into three other cell types, R-cells, F-cells and B-cells, each of which have different absorptive and secretory roles in the biology of the overall organ. This investigation uses centrifugal elutriation to separate the individual hepatopancreatic epithelial cell types of Homarus americanus and to investigate their plasma membrane copper transport properties using the copper-sensitive fluorescent dye Phen Green. Results show highly dissimilar endogenous concentrations of copper in each cell type and within the vacuoles (vesicles) released from these cells during the centrifugation process ([copper] in vacuoles>E-cells>R-cells>F-cells approximately B-cells). All four cell types were able to absorb copper from external concentrations ranging from 0.01 to 8 micromol l(-1), but considerable differences in transport rates occurred between the cell types. External calcium (0--10 mmol l(-1)) stimulated the uptake of external copper in a saturable fashion, suggesting the occurrence of carrier-mediated metal uptake. Addition of the Ca(2+) channel blocker verapamil (30 micromol l(-1)) to the external medium reduced the uptake rate of copper by all four cell types, but to different extents in each type of cell. External zinc (0--1000 nmol l(-1)) was a competitive inhibitor of copper influx in E- and R-cells, suggesting that the two metals shared the same binding and transport mechanism. A model is proposed which suggests that copper may enter all hepatopancreatic epithelial cell types by a divalent cation antiport process that exchanges intracellular Ca(2+) (or other cations) with either external copper or zinc. Verapamil-sensitive Ca(2+) channels may allow access of external calcium to cytoplasmic exchange sites on the antiporter or to activator sites on the same transport protein. The results suggest that elutriation is an excellent technique for the separation of complex invertebrate organ systems into their separate cell types and for analyzing the physiological properties of each cell type in isolation.

Animals↗