Isolation of motile spermatozoa by density gradient centrifugation in Percoll®

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.     

Separation of Non-filamentous Micro-organisms from Soil by Density Gradient Centrifugation in Percoll

Soil suspensions were homogenized, and desorbed non-filamentous micro-organisms were concentrated in a minimum volume of buffer by low speed centrifugation. The cells were separated from inanimate material by flotation at the interface between the buffer and a silica sol/polyvinyl pyrrolidone density gradient medium (Percoll). Cell suspensions were removed from the interface and fractionated according to density by high speed centrifugation on discriminating density gradients in Percoll.
Preliminary experiments indicated that most non-filamentous soil micro-organisms had densities in the range 1.081–1.123 g%sol;ml while Rhizobium isolated from crushed root nodules on Percoll was split into two bands of densities 1.081–1.110 and 1.041–1.073 g/ml. The lighter cells were the more pleomorphic.
The efficiency of extraction of cells from soil was governed by the extent of their desorption from inanimate particles. As rigorous desorption procedures damage cells, extraction efficiencies were low; 10–20% of cells counted microscopically in soil were recovered from density gradients. Electron microscopy of soil micro-organisms isolated by this method showed an unusual range of surface ornamentations on cell-like structures of bacterial dimensions.     

Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P₃) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P₃ were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P₃ was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed-angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm³ while those containing NaK ATPase were found at 1.047 g/cm³. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm³ suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ-glutamyl transpeptidase (γ-GTP); this separation was most pronounced in 12% Percoll. The activity of γ-GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane-bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ-GTP activity of choroid plexus, γ-GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.     

Classification of the isolated hepatocytes

At this present, enzyme perfusion method is a routine technique to isolate hepatocytes from rat liver for the physiological and pathological experiments. This study described a way of the classification of freshly isolated hepatocytes. First of all, the hepatocytes were fractionated with parenchymal and non-parenchymal cells by low speed centrifugation. And then these cells were subfractionated with a newly developed Percoll linear density gradient method. The fractionated parenchymal cells were divided with cells of periportal and centrilobular areas, respectively. Furthermore, their characteristics were confirmed functionally and morphologically. Non-parenchymal cells (NPC) include Kupffer cells, endothelial cells and fat storing cells (FSC, Ito cells). These isolated NPC are fractionated with a method as mentioned above or centrifugal alutriation method. In this paper, fractionation and classification of Kupffer cells and FSC were discussed with the measurement of fluorescent intensity of vitamin A and the morphological observation of cytoskeleton in culture. Especially, transport of vitamin A into FSC were detected autoradiographically.     

Functional testing of hepatocytes following their recovery from cryopreservation

Various tests of function have been suggested for assessing hepatocytes recovered from cryopreservation. In this study we have investigated hepatocyte attachment during tissue culture and cellular density in order to assess function and compared them with two classical dye exposure tests. The ability of hepatocytes to exclude trypan blue dye (TB) and metabolize fluorescein diacetate (FDA) was demonstrated. In populations of freshly prepared hepatocytes 88.07% were able to exclude TB and 87.31% were able to metabolize FDA. However in populations of hepatocytes recovered after cryopreservation using 1.5 M dimethyl sulfoxide as cryoprotectant only 33.44% were able to exclude TB and 31.59% able to metabolize FDA. Both of these tests gave the same estimate of functional ability. Density gradient centrifugation of hepatocytes on Percoll 400 (Pharmacia, Uppsala, Sweden) separated two populations of hepatocytes; one (density ca.1.07 g/ml Percoll) in which most of the cells were able to exclude TB and the second (density ca. 1.02 g/ml Percoll) in which they were stained blue. The dense population was highly enriched in dye-excluding hepatocytes: freshly prepared hepatocytes, 92.4%, and cryopreserved hepatocytes, 88.66%. When samples of these cells (2 x 10(6) dye-excluding cells per dish) were tested for their ability to attach to tissue culture dishes only 17.28% of the cryopreserved hepatocytes were able to attach compared to 55.28% of the freshly prepared cells. We conclude that cryopreservation of hepatocytes produces a population of cells which are not metabolically identical to a population of freshly prepared hepatocytes even though they appear to have the same buoyant density and dye-excluding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of spinach leaf peroxisomes in 0.25 molar sucrose solution by percoll density gradient centrifugation

A procedure for isolating spinach (Spinacia oleracea L.) leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation followed by removal of the Percoll by washing and centrifugation was established. The preparation contains more than 90% peroxisomes as intact organelles with no detectable chlorophyll or cytochrome oxidase contamination. The peroxisomes are stable at 0 to 4°C or 25°C for at least 2 hours.     

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.     

Fractionation of cells and subcellular particles with Percoll

At present, centrifugation is the most common method for separation and isolation of cells and subcellular particles. The technique can be used for a wide range of applications. During latter years it has become obvious what a powerful method density gradient centrifugation is, especially when used in conjunction with sensitive assays or clinical treatments. The most active areas for use of density gradient centrifugation include purification for in vitro fertilization of sperm of both human and bovine origin, isolation of cells for cell therapy of patients receiving chemo- and radiation therapy and basic research both on cellular and subcellular levels. These treatments and investigations require homogeneous populations of cells and cell organelles, which are undamaged after the separation procedure. Percoll, once introduced to reduce convection during centrifugation, has proved to be the density gradient medium of choice since it fulfills almost all criteria of an ideal density gradient medium. Recently good results have also been obtained after silanization of colloidal silica particles, e.g. BactXtractor. The latter medium has proved to be useful in recovery of microorganisms from food samples free of inhibitors to the Polymer Chain Reaction (PCR). The separation procedures described for Percoll in this review seem to be applicable to any cells or organelles in suspension for which differences in size or bouyant density exist. Furthermore, since Percoll media are inert, they are well suited for the separation of fragile elements like enveloped viruses.     

A practical method of Ludox density gradient centrifugation combined with protargol staining for extracting and estimating ciliates in marine sediments

Methodological impediments have long been the main problem in estimating the ecological role of marine benthic ciliates. Percoll density centrifugation is currently the most efficient technique for extracting ciliates from fine-grained sediments, while the high cost and low density of Percoll limit its wide application. We developed a protocol of density gradient centrifugation using the cheap sol Ludox HS 40 in combination with the quantitative protargol stain (QPS) to enumerate and identify marine benthic ciliates. The combined Ludox-QPS method involves sample collection and salt reduction, extraction with Ludox centrifugation, and preparation with the QPS technique. The recovery efficiency of Ludox was first tested with azoic sandy and muddy sediments. A 94–100% recovery rate of ciliates was reached. The method was further tested with natural sandy, muddy-sand and muddy sediments. Excellent extraction efficiencies were consistently obtained: an average of 97.6% for ciliates in sand, and 96.9–97.8% for nematodes in the three types of sediments. The high efficiencies indicate that the method allows for simultaneous enumeration of micro- and meiobenthos. Advantages of the new method include: (i) reliable and cost-efficient operation; (ii) appropriate centrifugation for both micro- and meiobenthos; and (iii) applicability to large samples and routine ecological surveys.     

Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes

Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6 M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6 M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19 kDa and two additional polypeptides of 23 and 26 kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum.     

A fast centrifuge method for nematocyst isolation from Pelagia noctiluca Forskal (Cnidaria: Scyphozoa).

Nematocyst isolation from surrounding tissue is an important step to characterize Cnidarian venom. Although several protocols have been used to extract venoms from cnidarian tissues, the complete isolation of nematocysts from tissue is still difficult. The goal of the present work was to evaluate the effectiveness of three different media, Percoll, Ficoll and Methylcellulose in isolating nematocysts from Pelagia noctiluca tentacles by centrifugation. The complete sedimentation of nematocysts and tissue fragments to the bottom of the test tubes was observed in Ficoll and Methylcellulose suspensions. The best result was obtained using a discontinuous density gradient of Percoll: three types of nematocysts were concentrated in three different fractions along the density gradient. Protein assay and preliminary chromatographic analyses confirmed these results.     

Purification of autophagosomes from rat hepatocytes

To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.     

Functional and physical characteristics of rat Leydig cell populations isolated by metrizamide and Percoll gradient centrifugation

The physical and functional properties of Leydig cell populations obtained by centrifugation of testicular cells in two different density gradient media, Percoll and Metrizamide, were compared. Percoll-gradient centrifugation yielded two Leydig cell bands (Peak I and Peak II) that were comparable, as to their density and testosterone-producing capacity, to the respective Leydig cell bands, Population I and Population II, isolated in a Metrizamide gradient. The denser Leydig cell band (II) had a greater capacity for testosterone production than the less dense band (I), regardless of the type of gradient used for its isolation. Metrizamide gradient centrifugation separated the majority of germ cells from the "light" (Population I) Leydig cells, whereas in the Percoll gradient, germ cells comigrated with Peak I Leydig cells. Leydig cell separation by Percoll gradients was highly dependent on the presence of Ca2+ and Mg2+ in the medium, while these cations had no effect on the separation of Leydig cells by Metrizamide. In conclusion, Metrizamide gradient centrifugation yielded two Leydig cell populations of similar functional and physical properties to the respective populations isolated in Percoll gradients.     

Nuclear buoyant density determination and the purification and characterization of wild-type neurospora nuclei using percoll density gradients

A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Observations on the separation of Theileria sporozoites from ticks

Hyalomma anatolicum anatolicum ticks infected with Theileria annulata were partially fed on rabbits and then ground up with tissue culture medium. The ground up ticks were treated by centrifugation at 100 g, filtration through membranes of 8 μm pore diameter and centrifugation on a discontinuous density gradient of Percoll. Counts of sporozoites and tick debris were made from Giemsa stained slides of samples at each stage of the separation. Debris was removed during light centrifugation and filtration at a greater rate than sporozoites. After filtration approximately 41% of the original sporozoites remained in the suspension. After density gradient centrifugation most sporozoites were found in a distinct zone, at approx. 1·08 g/cm³ density, separate from most dense debris and light debris and soluble contaminants. After this final centrifugation approximately 24% of the original sporozoites remained in the recovered suspension.     

Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 +/- 12-fold (mean +/- standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mM methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both beta-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.     

Evaluation of separating X- and Y-sperms by percoll density gradient centrifugation using sperms of transgenic mice carrying a transgene on Y-chromosome

Using sperms of the transgenic mice carrying a human A gamma/beta-globin gene on Y-chromosome, we attempted to separate X- and Y-bearing sperms by the Percoll density gradient centrifugation. The ratio of X- and Y-sperms was determined by DNA dot blot hybridization procedure with sperm DNA. Sperm suspension collected from cauda epididymidis was loaded on the gradient composed of 7 Percoll concentrations (35-84%) and was centrifuged at 300 x g for 10, 15 or 20 minutes, respectively, at room temperature. After centrifugation, sperms were collected from each gradient fraction and washed with 0.85% saline solution. DNA was extracted from sperms, dotted and fixed on nitrocellulose filter, and was hybridized with the 32P-labeled DNA probe derived from the beta-globin gene. Each DNA spot was cut out, immersed in the liquid scintillator and was counted for radioactivity. There was no difference among the radioactivities in the DNA spots, indicating that the ratio of X- and Y-sperms was the same in all the gradient fractions of three different centrifugal conditions. The results suggests to be difficult to separate X- and Y-sperms by Percoll density gradient centrifugation, at least, using sperms from cauda epididymidis of mouse.     

The isolation of intact adrenal chromaffin granules using isotonic Percoll density gradients

An improved method for the isolation of intact adrenal chromaffin granules under isotonic conditions, using a Percoll density gradient, is presented. After dissection, homogenization, and differential centrifugation, the crude granule homogenate was layered onto a gradient medium previously centrifuged at 20,200g × 5 min, consisting of 30% ($\text{v}\text{v}$) Percoll, 0.27 m sucrose, and 10 mm Tris-maleate, pH 7.0. After centrifugation for 40 min at 8650g in a standard preparative centrifuge, the chromaffin granules were found to band in the lowest fraction, where 45% of the catecholamines and 60% of the ATP could be recovered. With respect to other published methods, the percentage of lysosomal and mitochondrial contamination compared favorably. In addition, granules isolated by the Percoll gradient method were found to have at least 42 and 14% higher ATP and catecholamines, respectively, per milligram of protein. It is suggested that this method offers the advantages of ease of preparation, purity, and cost efficiency when compared with previously published techniques.