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PubMed1984

Purification of the scrapie agent by density gradient centrifugation.

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with 125I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30 000. No differences were seen between proteins in healthy or scrapie-infected preparations.

Animals↗
PubMed1992

RNA isolation from cartilage using density gradient centrifugation in cesium trifluoroacetate: an RNA preparation technique effective in the presence of high proteoglycan content.

An efficient method for the isolation of RNA from cartilage is described. The difficulties in obtaining RNA from cartilage, a tissue of low cell density and high proteoglycan content, were overcome by making several modifications to the guanidine thiocyanate/cesium chloride method of RNA extraction. Cartilage tissue is frozen, crushed, and homogenized in a 4 M guanidine thiocyanate lysis buffer. The RNA is then pelleted by ultracentrifugation through a cesium trifluoroacetate density gradient. The use of cesium trifluoroacetate, rather than cesium chloride, for density gradient centrifugation improves both the yield and purity of total RNA isolated from cartilage. The ultracentrifugation has been adapted to the Beckman TL100 tabletop centrifuge and is complete in 3 h. This fast, simple method produces high quality RNA, suitable for use in RNase protection assays, polymerase chain reaction analysis, and Northern analysis. This purification procedure may be applicable to other sources, from which RNA isolation is complicated by the presence of abundant cell wall or matrix components.

Animals↗
PubMed1984

Equilibrium density gradient centrifugation of the scrapie agent in Nycodenz.

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains were detergent-extracted, then separated by equilibrium density centrifugation in continuous Nycodenz gradients. The highest level of infectivity was always associated with the insoluble residue which sedimented through 40% Nycodenz. The degree of aggregation in these insoluble complexes varied depending upon treatment. Centrifugation in gradients containing 2 M- to 8 M-urea resulted in the formation of large insoluble aggregates which seemed to retain a high level of infectivity when measured by the method of incubation interval assay. However, measurement of infectivity in these same samples by endpoint titration of tenfold dilutions resulted in values a thousand times lower. These observations reinforce previous findings that scrapie infectivity exists as a macromolecular complex and, furthermore, they emphasize the necessity for using non-denaturing conditions for purification of the scrapie agent.

Animals↗
PubMedDate not supplied

Profiles of sperm morphology and motility after discontinuous multiple-step Percoll density gradient centrifugation.

Morphology and motility are important parameters for assessing the fertilizing capacity of spermatozoa. This investigation reports a systematic study on the profiles of these parameters after percoll gradient centrifugation. Spermatozoa from normal human semen were fractionated by discontinuous Percoll gradients (30%, 45%, 75%, 90%). Spermatozoa washed with Earle's balanced salt solution were used as a control. After centrifugation, sperm morphology was evaluated according to strict criteria; motility was assessed by a computer-assisted semen analysis system. The results showed that the percentage of spermatozoa with normal morphology increased, while those with severe morphological defects decreased, as the density of Percoll increased. The percentages of motile spermatozoa and hyperactivated spermatozoa, and the velocity and amplitude of lateral head displacement of the spermatozoa were significantly higher in the 75% and 90% Percoll fractions than that in the 30% and 45% ones, and in the control. These results demonstrated that Percoll density gradient centrifugation enriched spermatozoa in terms of morphology and motility.

Cell Separation↗
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↗
PubMed1985

Purification of thyroid lysosomes by colloidal silica density gradient centrifugation.

A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.

Acid Phosphatase↗
PubMed1987

Human peripheral blood and bone marrow cell separation using density gradient centrifugation on Lymphoprep and Percoll in haematological diseases.

Density gradient centrifugation on Lymphoprep as described by Böyum [1] was used for mononuclear cell separation of peripheral blood (PB) and bone marrow (BM) samples in healthy subjects and in haematological diseases. The cells were analysed morphologically from cytocentrifuge preparations stained with May-Grünwald-Giemsa. At least 200 cells per preparation were counted manually. The separation results of PB samples in healthy subjects were comparable with those described by Böyum. This method was also suitable for the separation of lymphocytes and blast cells in chronic lymphoproliferative diseases and in acute leukaemias, respectively. In other haematological diseases, however, the mononuclear cell fractions of PB samples and especially of BM samples were contaminated with myeloid and erythroid cells. Percoll gradients were used with success for the enrichment of myeloid BM cells and PB reticulocytes. However, absolutely pure myeloid cell fractions from different maturation stages could not be separated. The results indicate that cells to be investigated should be morphologically examined, to avoid erroneous interpretation of biochemical and functional activity of the cells.

Blood Cells↗
PubMed1988

Erythrocyte carnitine: a study of erythrocyte fractions isolated by discontinuous density gradient centrifugation.

Erythrocyte fractions of varying density were isolated by discontinuous density gradient centrifugation of washed erythrocytes of five subjects (three adults and two cord bloods). Free and total carnitine concentrations were determined in each gradient fraction to compare the carnitine content of less dense with more dense erythrocytes. Erythrocyte, leukocyte, and reticulocyte counts and hemoglobin were measured on all fractions of each gradient. The density gradient studies showed that the highest proportion of reticulocytes were associated with the least dense gradient fractions of all five subjects. Linear regression analyses revealed significant positive correlations (r = 0.94 to 0.99, P less than 0.02 to P less than 0.001) between the number of reticulocytes per fraction and the total or free carnitine concentrations per fraction for all subjects. No correlation was found between free or total carnitine and hemoglobin, number of erythrocytes, or number of leukocytes per fraction. It appears that erythrocyte carnitine is localized in circulating reticulocytes which have mitochondria and carnitine-dependent fatty acid metabolism.

Adult↗
PubMed1987

Morphologic heterogeneity of cell populations isolated by density gradient centrifugation from serous fluids of ovarian tumors.

The cells of tumor fluid from patients with malignant and benign serous ovarian neoplasms were fractionated using Ficoll-Uropoline density gradient centrifugation. Density distribution and morphologic characteristics of cell fractions were analyzed. It was found that serous ovarian adenocarcinomas contained three to four types of morphologically malignant cells focused in low density layers. Borderline ovarian neoplasms showed the presence of one subpopulation of cells with some features of malignancy and cells with some atypical but non-malignant features. The fluids of serous cysts contained mainly normal epithelial cells representing different stages of morphological maturity and were focused in denser layers. The results allowed us to catalogue ovarian tumor cell subpopulations present in each density fraction of individual patients and confirmed that ovarian tumors could be diagnosed by morphologic identification of cells from tumor fluids.

Adult↗
PubMed1975

Augmentation of marrow growth by thymocytes separated by discontinuous albumin density-gradient centrifugation.

Mouse thymocytes were separated by discontinuous albumin density-gradient centrifugation. The ability of B6 thymocytes from the gradient fractions to increase the number of spleen nodules formed by B6 marrow cells in B6D2F1 recipients was compared with that of similar numbers of B6 thymocytes from an unfractionated cell suspension. Thymocytes from any of the gradient fractions were not more effective than thymocytes from an unfractionated suspension in augmenting marrow-cell growth, indicating that the presumptive 'effective cell type' in this system cannot be separated from the total thymocyte population by its buoyant density properties.

Animals↗
PubMed2001

Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception.

BACKGROUND: With an increase in the use of assisted reproduction technologies the requirements of the diagnostic semen analysis are constantly changing. METHODS: Spermatozoa from patients undergoing IVF were analysed by examining the conventional semen parameters and DNA/chromatin integrity, using in-situ nick translation (NT) and the Chromomycin A(3) fluorochrome, which indirectly demonstrates a decreased presence of protamine. Samples were examined before and after preparation using discontinuous density gradient centrifugation. RESULTS: Density gradient centrifugation enriched samples by improving the percentage of morphologically normal forms by 138% and sperm nuclear integrity by 450%. Sperm nuclear integrity as assessed by in-situ nick translation (NT) demonstrated a very clear relationship with sperm concentration, motility and morphology. Morphology correlated with fertilization rates of patients undergoing IVF, while NT values of the spermatozoa post-preparation were significantly lower in pregnant patients. CONCLUSIONS: We have demonstrated that along with the classical semen parameters, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating patients to specific assisted reproduction treatments.

Adult↗