Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

A new method designed for the specific isolation and characterization of ligand-receptor complexes using a heterobifunctional crosslinking agent and immunoprecipitation is described. The complexes are first covalently crosslinked by photoactivation of the crosslinking agent. After lysis of the cells, the crosslinked complexes are immunoprecipitated using an antiserum directed against the crosslinking agent. With this method, ligand-receptor complexes formed in only minute amounts become available for further investigation. By using this anticrosslinker antiserum, different receptor systems can be investigated without raising new receptor- or ligand-specific antibodies for each system. As a test system, a radioiodinated lectin was used as ligand molecule and erythrocyte membranes acted as receptor carriers.     

Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.     

Use of Percoll density gradient centrifugation for preparing isolated rat hepatocytes having long-term viability

A method for preparing suspensions of hepatocytes with long-term viability is described. Its originality lies in the use, after conventional preparation, of a Percoll density gradient centrifugation which allows a complete and rapid separation of cell debris and released proteases from the intact hepatocytes. A series of functional reactions characterizing drug metabolism (P-450 level, ethoxycoumarin hydroxylation, methylumbelliferone conjugation) and membrane transport and integrity (α-aminoisobutyric acid transport and its stimulation by both insulin and glucagon) were conducted, in parallel to the trypan blue exclusion test, to evaluate the metabolic activity of both conventional and Percoll preparations. This investigation clearly demonstrates that the use of Percoll density gradient centrifugation significantly increases (from 5–6 to 30–35 h) the half-life of freshly isolated hepatocyte suspensions.     

Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation

We investigated relationship between the maturity and density of muscle cells and developed a rapid isolation method to acquire stem cells from skeletal muscle. Mononuclear cells were isolated from the lower hind-limb muscles of 7-d-old male Sprague–Dawley rats and separated by Percoll density gradient centrifugation. After centrifugation, the cells were layered in the interfaces between each Percoll density layer. Flow cytometry was used to investigate the Sca-1, Pax7, CD34, CD45, M-cadherin, and myosin expression of the cells in each density layer. We found that CD45-positive cells were not present in freshly isolated muscle cells. CD34-, Pax7-positive cells were mainly observed at the interface between the 15% and 25% Percoll layers and had a density of 1.0235–1.0355 g/ml. Cells positive for M-cadherin were at the 25–35% Percoll density interface and had a density of 1.0355–1.0492 g/ml. We conclude that because there appears to be a correlation between maturity and density, muscle-derived stem cells may be isolated successfully from the 15–25% Percoll interface.     

Isolation of pea nodule bacteroids utilizing silica sol (Percoll) density gradient centrifugation

Isolation of bacteroids from effective (Fix⁺) and ineffective (Fix^–) pea nodules, inoculated withRhizobium leguminosarum K, were performed by a density gradient centrifugation method using silica sol (Percoll). Only one zone (=1.064–1.072; n-zone) was recognized in the Fix⁺ nodule which contained typical Y-shaped bacteroids while two zones (n-zone and =1.125–1.145; n'-zone) were obtained from the Fix^– nodule. The cells in the n'-zone, which are long rods differed morphologically from free-living cells at any growth phase (=1.108–1.125; f-zone and =1.074–1.078; f'-zone), and differed from Y-shaped bacteroids by cell density. The esterase isozyme pattern of bacteroids in the n-and n'-zones also showed clear differences from that of f-and f'-zone of free-living cells.     

Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.     

Purification of human sperm by a discontinuous Percoll density gradient with an innercolumn

Human sperm were highly purified through the use of a discontinuous Percoll density gradient placed in an inner column of a centrifuge tube. Six ml of 80% Percoll solution were poured into a centrifuge tube with an inner column containing successive 1.0-ml layers of 70, 60, and 40% Percoll solutions. Diluted semen was placed on top of the gradient, and the tube was centrifuged at 600 X g for 30 min using a swing-out rotor. After centrifugation, the majority of the progressive motile sperm were isolated in the sediment; they had a mean motility of 93 +/- 4.1% (n = 10). Other cellular components, including bacteria, remaining in the inner column. The level of bacterial contamination in the purified sperm fraction was below detection for most of the species quantified. The purified sperm were found to be more than 92 +/- 3.2% viable, as judged by dye exclusion, and abnormal sperm were reduced to 5.2 +/- 1.4%. Because of the use of the inner column, the contamination by seminal plasma was negligible in the purified sperm, as estimated by residual protein, fructose, and acid phosphatase activity.     

Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure

The selection of motile human spermatozoa, from fertile and infertile semen samples was compared by using Percoll density gradient centrifugation or the swim-up procedure. Selected spermatozoa were evaluated according to their motility, % normal forms, nuclear maturity (aniline blue staining, acridine orange staining, ethidium bromide uptake and SDS nuclear decondensation). These methods showed differences between fertile and infertile men. The swim-up procedure, based on motility, resulted in greater proportions of motile spermatozoa and eliminated mainly tail abnormalities. Percoll gradient separation, based on density, selected oval-headed spermatozoa with good motility. Nuclear maturity level was improved by both methods but Percoll gradient separation generally resulted in spermatozoa with better nuclear maturity than those selected by the swim-up procedure.     

Isolation of Kurloff cells by Percoll density gradient centrifugation. Protein labeling with 35S-methionine of these cells

Large populations of splenic Kurloff (150 - 200 X 10(6) Kurloff cells) were obtained from estrogenized guinea pigs by isopycnic centrifugation in a Percoll solution of 1.085 g/ml starting density. The Kurloff cells settled at a buoyant density of about 1.100 g/ml. The purity of these cell suspensions reached 95%, as assessed by phase contrast microscopy and by specific staining. The viability assessed by Trypan blue exclusion test was also about 95%. Moreover, the good transmission electron microscopic appearance of these Kurloff cells and their ability to take up 35S-methionine in culture confirmed their physiological integrity. By autohistoradiography, this protein labeling was localized between the nucleus and the Kurloff body, and also on the Kurloff body itself. This data reinforces the hypothesis of de novo synthesis of the Kurloff body.     

Comparison of the coverslip and the discontinuous Percoll density gradient methods of enucleation of mouse cells

Two methods of enucleation of LB 10 cells, a subline of mouse L cells, were used: the method of enucleation of cells growing in monolayers, and the newly improved method of enucleation in discontinuous Percoll gradients. The second method was more effective and, as shown by incorporation of 3H-lysine, protein synthesis in cytoplasts was prolonged twice when compared with that in cytoplasts obtained by the coverslip method.     

Characterization of rat epithelial epididymal cells purified on a discontinuous Percoll gradient.

A method for the purification of epithelial cells from the three anatomical regions of the rat epididymis (corpus, caput and cauda) is described. An enzymic digestion followed by sedimentation of crude cell suspension on discontinuous Percoll gradient yielded quite pure active epithelial cell population as judged by morphological and functional studies. Electron microscopy analysis showed that cells from bands corresponding to densities 1.055 and 1.06 g/ml the gradient preserved a morphology compatible with their epithelial origin and their absorptive and secretory functions. Moreover, they stained positively with anticytokeratin antibody (95-97%) and were negative for antidesmin antibody. They selectively bound L-carnitine through a time-dependent and saturable system and differences in the rate of binding were apparent according to the three anatomical regions of the epididymis.     

Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.     

Electrophoretic separation of cells in a density gradient.

A bulk electrophoretic method is described for the separation of mammalian cells in Ficoll-sucrose density gradients. The fractionation of cells is performed in a simple commercially available apparatus (Buchler Poly-Prep) which should facilitate the application of the technique in various laboratories. Details of the experimental procedure are presented along with typical model separations of erythrocytes from various species and mouse lymphocytes.     

Trypsin sensitivity of mammalian cells grown in a serum-free medium

Summary Two mammalian cell lines which multiply in vitro in culture medium devoid of serum were investigated for sensitivity to twice crystallized trypsin. The minimum trypsin concentration which showed a concomitant increase in cell numbers and detachment from the surface of the culture vessel in one cell line was 0.01 μg per ml or approximately 10⁻⁸ μg per cell. These effects could be neutralized by normal human or calf serum at a dilution of 1∶500. The second cell line, which normally grows in suspension, was unaffected by these concentrations of trypsin.     

Interferon production by mammalian cells grown in a serum-free medium

Summary Interferon, produced by rabbit heart cells grown in a serum-free medium, failed to protect rabbit heart serum-free cells, but protected rabbit heart serum-containing-medium cells against vaccinia and vesicular stomatitis virus. Interferon produced in serum-free cells had a greater species specificity than that produced in serum-containing media. The difference in activity was shown to be due to lack of adsorption by serum-free-medium cells.     

Isolation of pancreatic autophagic vacuoles induced by vinblastine or neutral red. Separation of autophagosomes and autolysosomes by Percoll density gradient centrifugation

Our purpose was to elaborate a cell fractionation method for the preparation and purification of macroautophagic vacuoles (AVs) and their subfractions: autophagosomes and autolysosomes. To overcome the difficulties caused in liver and some other cell types by the overlapping buoyant densities and sizes of different subclasses of lysosomes and other subcellular particles, we chose the murine pancreatic acinar cell as experimental system in which enormous numbers of large-sized AVs are readily accumulated upon certain treatments. As we measured by electron microscopic morphometry, cytoplasmic volume fraction of AVs was as small as 0.31% in the untreated cells, while it was elevated to 8.1% 4 h after the injection of 50 mg/kg body weight vinblastine sulfate (a widely used inducer of macroautophagy). From vinblastine-treated pancreas, a 5500g sediment containing AVs and mitochondria (AV-M fraction) was obtained by differential centrifugation. This fraction was resolved in a 50% Percoll gradient (15 min, 92,000g) into three distinct particle populations. Mitochondria were localized near the upper boundary of the gradient at a buoyant density of 1.075 to 1.08, whereas directly under them light AVs (1.085-1.09) were banded. Heavy AVs (1.13-1.14) formed a broad layer near the bottom of the tube. Electron microscopic comparison of the morphology of these fractions and AVs in situ showed that light AVs correspond to AVs in early, whereas heavy AVs to AVs in advanced and late stages of degradation of the segregated material. The activity of lysosomal enzymes were found low in both fractions, being several times higher in the heavy than in the light one.(ABSTRACT TRUNCATED AT 250 WORDS)