Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

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.     

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.     

Adenosine triphosphate content of Mycobacterium leprae by percoll buoyant density centrifugation.

Thirty nine untreated patients of bacilliferous leprosy with a mean bacteriological index of 4.8 and morphological index of 1.3% formed the study group. Adenosine triphosphate assay was carried out by (i) enzyme treatment method in 18 patients and (ii) percoll buoyant density gradient method in 21 patients. ATP content obtained by percoll buoyant density gradient method was significantly higher than that obtained by enzyme treatment method. Percoll buoyant density centrifugation for purification and isolation of bacilli from human leproma is simplier, quicker and can serve as an alternate method of enzyme treatment.     

Isolation of a purified mitochondrial fraction from viable clonal insulin-producing cells (RINm5F) by percoll density gradient centrifugation

Summary A Percoll density gradient was employed for selecting large numbers of viable insulin-producing RINm5F cells. Homogenates of these cells were then subjected to gradient centrifugation and two clearly visible bands were obtained. The light fraction was essentially composed of mitochondria banded at a density of about 1.06 g/ml. The heavier fraction banded at 1.09 to 1.10 g/ml and contained lysosomes and a small number of secretory granules. The distribution of Percoll particles was restricted to the extracellular space and there was no adsorption to any membrane structures. The distribution pattern of marker enzymes for the mitochondria and lysosomes was similar to that of normal pancreatic β-cells. With the use of a Percoll density gradient it was thus possible to isolate a purified mitochondrial fraction from viable RINm5F cells. This work was supported by the Swedish Medical Research Council (03x-4, 12x-562, 12x-6240), the Swedish Diabetes Association, the Nordic Insulin Foundation, Syskonen Svenssons Foundation, and ?ke Wiberg’s Foundation. Per-Olof Berggren is a recipient of a postdoctoral fellowship from the Swedish Medical Research Council.     

Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Evaluation of centrifugation parameters for density gradient experiments by means of a programmable pocket calculator

A calculation program is proposed suitable for programmable pocket calculators (e.g. HP series) to estimate s_20,w ∫ ω² dt values from density gradient centrifugation data. The program can be applied to linear or exponential density gradients prepared from sucrose or glycerol solutions spun in zonal rotors or swinging bucket rotors. A wide solute concentration range and temperature range is accounted for. Constants for empirical density calculation of glycerol and sucrose solutions concentrated in % (w/v) are estimated. Experiment verification of the program was carried out.     

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.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )     

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 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 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.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

Summary A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Purification of bovine neurosecretory granule membranes by density gradient centrifugation

A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretory granule fraction and were highly enriched in cytochrome b561. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content.