scholarly journals Is Percoll innocuous to cells?

1982 ◽  
Vol 202 (3) ◽  
pp. 795-797 ◽  
Author(s):  
J S Wakefield ◽  
J S J Gale ◽  
M V Berridge ◽  
T W Jordan ◽  
H C Ford
Keyword(s):  
Cell Surface ◽  
Leydig Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Peritoneal Macrophages ◽  
Isolated Rat Liver ◽  
Subcellular Organelles ◽  
Large Numbers ◽  
Gradient Medium ◽  
Isolated Rat

Peritoneal macrophages from mice, isolated rat liver Kupffer cells and rat testis Leydig cells ingested large numbers of Percoll particles, a gradient medium widely used for separation of cells and subcellular organelles by density-gradient centrifugation. A decrease in the percentage of macrophages adhering to plastic also occurred after exposure of the cells to Percoll, even at 4 degrees C, a temperature at which Percoll was not ingested. The effect of Percoll on macrophage adherence may involve a loose association between the density medium and the cell surface. Other cell-surface-related phenomena may also be affected by prior exposure of cells to Percoll.

Separation and characterization of Leydig cells and macrophages from rat testes

1991 ◽  
Vol 130 (3) ◽  
pp. 357-365 ◽  
Author(s):  
G. Dirami ◽  
L. W. Poulter ◽  
B. A. Cooke
Keyword(s):  
Monoclonal Antibody ◽  
Density Gradient ◽  
Leydig Cells ◽  
Magnetic Beads ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Average Density ◽  
Centrifugal Elutriation ◽  
Percoll Gradients

ABSTRACT A method involving centrifugal elutriation followed by density gradient centrifugation and incubation with a macrophage monoclonal antibody has been investigated to separate and characterize Leydig cells and macrophages from adult rat testes. After dispersion of the testes with collagenase, the isolated interstitial cells were found to contain 18% Leydig cells and 12% macrophages. These cells were then separated by centrifugal elutriation into eight fractions (F1–F8) (9 to 74 ml/min at 386 g). Each of these fractions was then further purified by density gradient centrifugation on 0–90% Percoll gradients. After centrifugal elutriation, the macrophages were mainly eluted in the first three fractions (F1–F3), whereas the Leydig cell percentage increased in each fraction with increasing flow rate. After further purification of each fraction on Percoll gradients, high percentages of macrophages (11–20%) were found in fractions F1–F3 (average density 1·045 g/ml), containing 11–37% Leydig cells. Less than 3% of the cells in fraction F4–F8 (average density 1 ·075 g/ml) were macrophages and more than 95% were Leydig cells. Heterogeneity of Leydig cells with respect to sedimentation velocities and function was found. Leydig cells from elutriated-and Percoll-purified fractions F4–F8 were heterogeneous with respect to testosterone and cyclic AMP (cAMP) production but showed a similar binding capacity for 125I-labelled human chorionic gonadotrophin. Leydig cells with the highest sedimentation velocity (35·7 mm/h-g) from fractions F7 and F8 were approximately twofold more responsive to LH (3·3 nmol/l) with respect to testosterone and cAMP production compared with Leydig cells with the lowest sedimentation velocity (20·7 mm/h-g). The elutriated and Percoll-purified cells (corresponding to fractions F4–F8) were further purified by incubation with magnetic beads coated with a macrophage monoclonal antibody; this yielded very pure Leydig cells containing <0·3% macrophages. The incubation temperature (room temperature or 4 °C) during the purification with magnetic beads did not affect the degree of purity or the responsiveness of the Leydig cells to LH. The removal of the remaining macrophages with magnetic beads did not have any significant effect on the Leydig cell responsiveness to LH. It was concluded that Leydig cells purified by elutriation and density gradient centrifugation are heterogeneous with respect to their sedimentation velocities and responses to LH; the higher the sedimentation velocity, the higher is their capacity to respond to LH. Leydig cells free from macrophages can be prepared by further purification using magnetic beads coated with a macrophage monoclonal antibody. Journal of Endocrinology (1991) 130, 357–365

A dolichol acyltransferase present in rat and human postheparin plasma

1992 ◽  
Vol 70 (6) ◽  
pp. 470-474 ◽  
Author(s):  
P. Sindelar ◽  
C. Valtersson
Keyword(s):  
Cell Surface ◽  
Molecular Mass ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
High Molecular Mass ◽  
Gel Chromatography ◽  
Heat Treated ◽  
A Cell ◽  
Small Unilamellar Vesicles ◽  
Plasma Heparin

Incubation of small unilamellar vesicles consisting of dioleoyl phosphatidylcholine – dioleoyl phosphatidylethanolamine (3:1) and 2 mol% [3H]dolichol-19 with postheparin plasma from rat resulted in the formation of dolichyl oleate. Normal plasma or heat-treated postheparin plasma contained no activity and, hence, the results indicate the presence of a cell surface associated dolichol acyltransferase that can be released into the blood by heparin. The reaction is strongly stimulated by phosphatidylethanolamine and Ca2+, whereas no stimulation with triglycerides or acyl-CoA was observed. Together with the fact that the only product formed was dolichyl oleate, these results strongly suggest that a transacylation mechanism from the phospholipids to dolichol is operative in the liposomes. Gel chromatography of postheparin plasma yielded a molecular mass of about 350 kilodaltons for the active enzyme and density gradient centrifugation indicated that this high molecular mass complex consists mainly of proteins. Finally, we conclude that this enzyme is not unique to the rat, but is also present in human postheparin plasma.Key words: phospholipids, dolichol, plasma, heparin, acyltransferase(s).

ISOLATION OF RAT LEYDIG CELLS BY DENSITY GRADIENT CENTRIFUGATION

1982 ◽  
Vol 92 (2) ◽  
pp. 293-NP ◽  
Author(s):  
J. S. GALE ◽  
J. ST J. WAKEFIELD ◽  
H. C. FORD
Keyword(s):  
Density Gradient ◽  
Binding Sites ◽  
Leydig Cells ◽  
Interstitial Cell ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Collagenase Treatment

A rapid method for preparing Leydig cells from rat testes is described. An interstitial cell suspension, prepared by collagenase treatment of decapsulated testes, was centrifuged for 10 min over a cushion of 60% (v/v) Percoll to remove red blood cells, and then centrifuged for 20 min in a 0–60% linear density gradient of Percoll. Seventy-four per cent of the cells present in that fraction of the gradient comprising 35–50% Percoll were Leydig cells; the yield from each testis was about 1·5 × 106 cells. The Leydig cells appeared viable, excluded Trypan blue, possessed high-affinity binding sites for human chorionic gonadotrophin (hCG) and synthesized increased quantities of testosterone in response to hCG. The cells could be stored overnight in 20% (v/v) glycerol at −20 °C, with only minimal effect on the specific activities of a number of enzymes used as markers of subcellular components. Testosterone production in vitro by the cells after storage for 20 h was greater than that of hCG-stimulated fresh cells and was not further increased by hCG.

ISOLATION OF HIGHLY PURIFIED LEYDIG CELLS BY DENSITY GRADIENT CENTRIFUGATION

Endocrinology ◽  
1977 ◽  
Vol 101 (2) ◽  
pp. 639-642 ◽  
Author(s):  
P. Michael Conn ◽  
Tsuneo Tsuruhara ◽  
Maria Dufau ◽  
Kevin J. Catt
Keyword(s):  
Density Gradient ◽  
Leydig Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation

Analytical Subcellular Fractionation of Guinea-Pig Myocardium

1977 ◽  
Vol 53 (1) ◽  
pp. 63-74
Author(s):  
F. J. Bloomfield ◽  
G. Wells ◽  
E. Welman ◽  
T. J. Peters
Keyword(s):  
Guinea Pig ◽  
Adenosine Triphosphatase ◽  
Small Volume ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Marker Enzyme ◽  
Subcellular Organelles ◽  
Dual Localization ◽  
Glutamyl Transpeptidase

1. Homogenates of guinea-pig left ventricle were fractionated by differential pelleting and by centrifugation on continuous sucrose density gradients. 2. The principal subcellular organelles of myocardium, characterized by their marker enzyme content, were resolved by density gradient centrifugation in a small-volume zonal rotor. The equilibrium densities (p) of the principal organelles are (with marker enzymes in parentheses): sarcolemma, 1·12 (5′-nucleotidase); lysosomes, 1·16 (N-acetyl-β-glucosaminidase); mitochondria, 1·17 (cytochrome oxidase); peroxisomes, 1·18 (catalase); cytosol (lactate dehydrogenase). 3. The subcellular distribution of various adenosine triphosphatase activities and previously unassigned enzymes was determined. Leucyl-β-naphthylamidase and γ-glutamyl transpeptidase showed both cytosol and sarcolemma components. Ca2+-dependent adenosine triphosphatase showed dual localization to the mitochondria and to the sarcolemma.

Rat parietal cell receptors for GLP-1-(7-36) amide: northern blot, cross-linking, and radioligand binding

1994 ◽  
Vol 267 (3) ◽  
pp. G423-G432 ◽  
Author(s):  
J. Schmidtler ◽  
K. Dehne ◽  
H. D. Allescher ◽  
V. Schusdziarra ◽  
M. Classen ◽  
...  
Keyword(s):  
Parietal Cell ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Parietal Cells ◽  
Small Cells ◽  
Cross Linking ◽  
Cell Content ◽  
Chief Cells ◽  
High Affinity ◽  
Isolated Rat

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal cells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isolated rat gastric mucosal cells (F0) were fractionated by counterflow elutriation, resulting in five fractions (F1-F5) according to increasing cell diameter and parietal cell content (3, 5, 4, 27, 81%). Additional density gradient centrifugation of F4 yielded enriched chief cells (74%; parietal cells: 1%; F6), whereas density gradient centrifugation of F5 almost purified parietal cells (97%; chief cells: 1%; F7). Northern blot of total cellular RNA from F0-F7 with a probe specific for the GLP-1-(7-36) amide receptor revealed two RNA species of 2.7 and 3.6 kb. These messages were present to some extent in small cells (F1, F2), much more pronounced in F5, abundant in F7, barely detectable in F3 and F4, and absent from F6. Cross-linking of 125I-labeled GLP-1-(7-36) amide to parietal cell membranes revealed a single 59-kDa band that was abolished by unlabeled GLP-1-(7-36) amide. Throughout fractions F1-F7 specific binding of 125I-GLP-1-(7-36) amide was correlated with parietal cell content (r = 0.99; P < 0.01) and H+ production ([14C]aminopyrine accumulation) in response to GLP-1-(7-36) amide or histamine (r = 0.96; P < 0.01). Binding was maximal in purified parietal cells (F7), whereas almost no binding was detectable in enriched chief cells (F6). In F7, Scatchard analysis revealed a single class of high-affinity binding sites (KD = 2.8 +/- 0.6 x 10(-10) M, Bmax = 6.8 +/- 1.4 fmol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The following half-maximal inhibition values were found for GLP-1-(7-36) amide and (1-37) and (1-36) amide: 6.6 +/- 0.9 x 10(-10), 1.4 +/- 0.7 x 10(-7), and 2.6 +/- 0.4 x 10(-7) M, respectively. Pancreatic glucagon, GLP-2, and oxyntomodulin, products of the proglucagon gene, were 3-4 log units less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that rat parietal cells are equipped with specific high-affinity receptors for GLP-1-(7-36) amide, which, in addition, are present in as yet unidentified small cells (F1, F2) but not in chief cells.

scholarly journals SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION

1972 ◽  
Vol 55 (3) ◽  
pp. 579-585 ◽  
Author(s):  
David A. Wolff ◽  
HÅkan Pertoft
Keyword(s):  
Density Gradient ◽  
Hela Cells ◽  
Colloidal Silica ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Conditions ◽  
Density Measurements ◽  
Interphase Cells ◽  
Gradient Medium ◽  
Vinblastine Sulfate

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.

Effects of arecoline on testosterone release in rats

2008 ◽  
Vol 295 (2) ◽  
pp. E497-E504 ◽  
Author(s):  
Shyi-Wu Wang ◽  
Guey-Shyang Hwang ◽  
Te-Jung Chen ◽  
Paulus S. Wang
Keyword(s):  
Anterior Pituitary ◽  
Leydig Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Male Rats ◽  
Time Intervals ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Steroidogenic Acute Regulatory

Arecoline is one of the major components of betel nuts, which have been consumed as chewing gum in Southeast Asia. In this study, the effects of arecoline on testosterone (T) secretion were explored. Male rats were injected with human chorionic gonadotropin (hCG, 5 IU/kg) or arecoline (1 μg/kg) plus hCG via a jugular catheter. Blood samples were collected at several time intervals subsequent to the challenge. Rat anterior pituitary was treated with gonadotropin-releasing hormone in vitro with or without arecoline, and then the concentration s of luteinizing hormone (LH) in the medium were measured. Rat Leydig cells were purified by Percoll density gradient centrifugation and incubated with arecoline, hCG, forskolin, 8-bromo-cAMP (8-Br-cAMP), nifedipine, nimodipine, or tetrandrine at 34°C for 1 h. A single intravenous injection of arecoline resulted in an increase of the hCG-induced level of plasma T. Administration of arecoline (10−8 to 10−6 M) in vitro increased T production in Leydig cells. The stimulatory effect of arecoline on T release in vitro was enhanced by hCG (0.001 IU/ml), forskolin (10−6 M), or 8-Br-cAMP (10−5 M). By contrast, nifedipine, nimodipine, or tetrandrine inhibited the increased T concentrations induced by arecoline. Western blot showed that arecoline increases steroidogenic acute regulatory (StAR) protein expression compared with vehicle. These results suggested that arecoline stimulates testosterone production by acting directly on Leydig cells via mechanisms involving an activation of L-type calcium channels, increasing the activity of 17β-hydroxysteroid dehydrogenase and enhancing the expression of StAR.

Sign in / Sign up

Export Citation Format

Share Document