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.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.     

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

Dopamine and serotonin in two populations of synaptosomes isolated by percoll gradient centrifugation

A technique has recently been developed for the isolation of synaptosomes by centrifugation through percoll gradients. Utilizing this procedure, striatal synaptosomes were separated into two fractions, termed fractions 3 and 4, by their different sedimentation characteristics in percoll. The aim of this investigation was to determine whether there were any neurotransmitter differences between these fractions. The content of endogenous neurotransmitters dopamine (DA) and serotonin (5-HT) significantly differed between these fractions. Fraction 3 contained greater levels of 5-HT, while fraction 4 was enriched for DA. Both fractions were capable of releasing DA or 5-HT upon K⁺ depolarization. The results raise the possibility that a relative enrichment of dopaminergic synaptosomes in fraction 4 and of serotonergic synaptosomes in fraction 3 has been achieved.     

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.     

Purification of metacyclic forms of Trypanosoma cruzi by Percoll discontinuous gradient centrifugation

A method for the purification of metacyclic forms of Trypanosoma cruzi has been developed. Metacyclic forms obtained in modified Grace medium were separated from the epimastigote forms by Percoll discontinuous density gradient centrifugation. Four different osmotic pressures were applied: 160 +/- 10, 260 +/- 10, 310 +/- 10 and 510 +/- 10 mosmol/kg H2O. At 160 mosmol/kg H2O, 100% of the metacyclic forms with a 21.3% yield were found in the interphase 1.120/1.125 g/ml, while 92.7% of the metacyclic forms with a 73.7% yield were found in the interphase 1.115/1.120 g/ml. At 310 mosmol/kg H2O, 100% of the metacyclic forms in the interphase 1.135/1.140 g/ml with a 36.8% yield were obtained. Metacyclic forms purified in this way do not show alterations in their capacity to infect cultures of HeLa cells.     

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.     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

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.     

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.     

Studies on thymocyte subpopulations in guinea pigs. Differences in cell volume and proliferative ability in vitro of two populations separated by density gradient centrifugation with Percoll

Thymus cells from guinea pigs were separated according to buoyant density by centrifugation with PVP-coated colloidal silica particles (Percoll). By creating an S-shaped density gradient, two populations (referred to as peak-I and peak-II cells) were obtained which differed in size as well as ability to spontaneous proliferation in vitro. Peak I contained low density cells of large size and was highly enriched with DNA-synthesizing cells. These continued to proliferate in culture for at least 30 h as demonstrated by mitotic studies in the intervals 0-10 and 20-30 h. In order to grow in vitro, however, the cycling cells of peak I depended on the medium (RPMI 1640) being supplemented with L-alanine. The high density cells of peak II constituted 70% of the thymocytes and had a small and uniform volume. This population was depleted of proliferating cells. The simple and rapid separation of these two major populations is considered a useful first step for the further characterization of thymocyte subpopulations. We suggest that peak I primarily includes proliferating precursor cells from the cortex as well as mature, immunocompetent cells. Peak II consists largely of small cortical cells.     

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.     

A rapid procedure for the isolation of lysosomes from kidney cortex by Percoll density gradient centrifugation

Highly pure lysosomes were isolated from buffalo(Bubalus bubalis) kidney cortex by a procedure involving differential and isopycnic Percoll density gradient centrifugations. Arylsulphatase, N-acetyl-Β-glucosamindase and cathepsin D in the lysosomal preparation were 26–45-fold enriched over the homogenate. The purified lysosomes contained less than 0·06% of mitochondrial, microsomal and peroxisomal marker enzymes. In the electron micrographs the particles appeared as large dense granules of size 0·3-1·9 μm with no apparent structural features belonging to mitochondria or microsomes. The isolation procedure was also found to be suitable to obtain highly pure lysosome particles from renal cortex of other sources such as rat, lamb and beef. No ultracentrifugation steps were involved in the procedure