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.     

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Percoll (colloidal silica coated with polyvinylpyrrolidone) and Ficoll (MW 400,000) were used to separate erythrocytes infected with Plasmodium yoelii and Plasmodium berghei from uninfected red blood cells. Samples of blood collected from mice in different phases of malarial infection were overlaid on cushions of 55% Percoll, 20% Ficoll, or 28% Ficoll, respectively, centrifuged, and the interphase layers compared. The best yield of parasitized erythrocytes (PE) was achieved using Percoll when about 95% of the erythrocytes infected by the late developmental forms of the parasites (late trophozoites, schizonts, and gametocytes) were recovered from the gradient interphase, irrespective of the phase of the infection and the number of young erythrocytes in the sample. No alteration of antigenicity (assessed by immunofluorescence) or of osmotic fragility (over the range of 160–460 mOsm) could be detected in PE separated by Percoll or by Ficoll. In addition, parasites separated on Percoll gradients showed no significant ultrastructural changes and retained their normal infectivity to mice. Although both gradient media could be used for the separation of Plasmodium-infected erythrocytes, Percoll presented some advantages over Ficoll. Apart from the better reproducibility of the separation of high yields of very pure PE obtained with Percoll, its lower viscosity allowed easier handling, and lower centrifugal forces were needed to enable the cells to reach their isopycnic positions. Thus, Percoll fulfilled many of the criteria for an ideal density gradient medium. Parasitized erythrocytes were isolated by an easy, reproducible, and inexpensive procedure, and separated cells retained their normal structure, antigenicity, and infectivity.     

Percoll and Ficoll self-generated density gradients by low-speed osmocentrifugation

Percoll and Ficoll self-generated density gradients can be obtained by low-speed centrifugation of their solutions within dialysis cells. Useful Percoll density gradients can be obtained after 10-30 min centrifugation at 220-2010g, within dialysis cells. Ficoll density gradients, which are more difficult to self-generate, can be obtained by the same technique. Red cell band formation in a Percoll density gradient can be done in a single step by using dialysis cells as the centrifugation solution container.     

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.     

Selective loss of chemokine receptor expression on leukocytes after cell isolation

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5 ± 2.9%) whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.     

Production of Ficoll, Percoll, and albumin gradients by the freeze-thaw method.

Ficoll, sucrose, albumin, and Percoll, a modified colloidal silica centrifugation medium, all form density gradients upon freezing and thawing. The data suggest that any density gradient material will form gradients upon freezing and thawing, provided the material is stable to freezing.     

Isolation of monkey aortic lysosomes using Percoll density gradient

A novel technique involving the Percoll density gradient and 0.01M phosphate buffer has been employed for the first time on aortic tissue for isolation of lysosomes. The purity of the lysosomes has been established by marker-enzymes, acid phosphatase and N-acetyl-beta-D-glucosaminidase and latent activities of lysosomal hydrolases. The heavier fraction (density 1.08) obtained after Percoll density gradient centrifugation showed high specific activities of lysosomal hydrolases and these enzymes were markedly latent. Moreover this heavier (lysosome rich) fraction has been noted to be free of other sub-cellular contaminants.     

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.     

Effects of castration and replacement of androgen on the separation of rat ventral prostate cells by Ficoll gradient centrifugation

When regressing or growing (hypertrophic) cells from collagenase-digested ventral prostates were centrifuged on isokinetic Ficoll gradients for 6-8 min, they distributed into four fractions. Because of changes in epithelial cell morphology and density following castration to induce regression and replacement of androgens to cause cell growth, and contrary to results with normal rat ventral prostate, stromal cell fraction 2 was contaminated to a greater extent with regressing epithelial cells, as judged by their morphology and binding of radioactive androgens. However, centrifugation for 3 min increased the purity of epithelial cell fraction 4, although the yield of desired cells was reduced. Most cells from endocrine-manipulated rats were viable, as judged by exclusion of trypan blue and the initial incorporation of 3H-uridine. Cells centrifuged on a similar gradient of Percoll separated by a 'sieving' effect, which inverted the order of cellular fractions and removed red blood cells from fraction 2. Metrizamide offered no advantages, compared with Ficoll or Percoll. Neither physiologic nor pharmacologic amounts of testosterone returned the morphology of isolated epithelial cells to normal. To obtain consistent results with prostates from normal or hormone-manipulated rats, one should take care to select an active preparation of collagenase, avoid the use of very old animals, cool the tissue after it is dissociated, and do not apply undigested clumps of cells or overload the gradient. If attention is paid to these details, populations enriched in viable regressing or growing prostate epithelial or stromal cells can be obtained from hormonally manipulated rats.     

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.     

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.     

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

Utilization of a test gradient enhances islet recovery from deceased donor pancreases

BACKGROUND: Islet transplantation is a viable treatment alternative for a select group of patients with type 1 diabetes. However, variables unique to the donor pancreas, such as age, fibrosis and edema, can influence the number and purity of the isolated islets. Thus isolation of a sufficient number of islets for transplantation from the pancreas remains challenging because of the lack of methods enabling reproducible isolation. METHODS: Islets were isolated from 38 consecutive deceased donors using the semi-automated Ricordi method of islet isolation, and purified on a COBE 2991 cell processor using Ficoll-based continuous density gradients. Three different gradient protocols were used. These included a pre-defined gradient using different densities of Ficoll (1.100 g/mL and 1.077 g/mL) mixed with HBSS (group 1), a pre-defined gradient using single-density Ficoll (1.100 g/mL) mixed with University of Wisconsin solution (UW) (group 2) and a variable gradient using single-density Ficoll (1.100 g/mL) with UW and densities selected based on the results of test gradients (group 3). RESULTS: Group 3 yielded a better recovery of islets (74%) than groups 1 (43%) or 2 (37%) (P=0.0144). Viability was significantly higher in groups 2 and 3 (P=0.0115). Purity was not significantly different among the groups. DISCUSSION: This method, using a simple test gradient, is a significant process improvement that can improve islet recovery without loss of viability or purity and increase the number of islet products suitable for transplantation.     

Improved purification of Piscirickettsia salmonis using Percoll gradients

Viable preparations of intact Piscirickettsia salmonis, purified from host cell material, are necessary for studying characteristics associated with this bacterium. However, purification of the organism is difficult due to its obligate intracellular nature. A simple and effective method for isolating whole P. salmonis, which is quick and easy to perform, but still maintains the viability and antigenicity of the bacterium is described. P. salmonis was purified by differential pelleting and density gradient centrifugation using 30%, 40%, or 50% (v/v) Percoll gradients. Following fractionation, a band with a density of 1.056-1.080 was found to be composed entirely of rickettsiae, confirmed by fluorescent antibody technique (IFAT). Purity of the P. salmonis from different stages of the purification process was assessed by light and transmission electron microscopy, and the viability of yields determined from a plaque assay and a tissue culture infective dose (TCID(50) ml(-1)). P. salmonis recovered from the 30% Percoll gradient appeared to retain their intracellular structure better than P. salmonis obtained from the 40% and 50% Percoll gradients, and appeared to have a greater viability. Differences were seen between P. salmonis-infected CHSE-214 cells and purified P. salmonis when compared by SDS-PAGE and Western blotting, and less host cell contamination was present in preparations obtained from the 30% Percoll gradient. Finally ten different P. salmonis isolates obtained from three different geographical locations and four different fish species, were purified using the 30% Percoll gradient. When the morphology of these was compared by transmission electron microscopy (TEM), they appeared similar in size and appearance, although isolate R980769 was highly pleomorphic and isolate R-29 was larger than the other isolates examined.     

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.     

Isopycnic centrifugation of antigen-activated human blood lymphocytes in the early phase of in vitro stimulation

The interrelationship was determined between osmolarity, concentration, and density of aqueous solutions of polysucrose (Ficoll), metrizoate (Isopaque), polyethylene glycol, gum acacia, and ioglycaminic acid (Bilivistan). It was found that by mixing two solutions—each consisting of different concentrations of Ficoll, Isopaque, Tris-HCl buffer, and a Ringer solution—a linear density gradient could be produced, ranging from 1.055 to 1.095 g/ml. This gradient has a constant osmolarity of 280–290 mOsm (isotonic with human serum) and a constant pH of 7.4. A method is described for the determination of density distribution profiles of human blood lymphocytes by means of this gradient. The procedure has a high reproducibility and does not change the lymphocyte viability as determined by subsequent multiplication in culture and dye exclusion. The mean density of human blood lymphocytes, immediately after isolation, was found to be 1.072±0.0014 (n = 12). The yield of lymphocytes from the linear gradient is 50–70 %, but can be increased to 80–90 % if 1 % human albumin is included in the gradient material. From a discontinuous gradient 80–90 % of the cells may be recovered. Moreover, it was found that the same method may be used for the determination of the density profile of human spleen cells, thymus cells, granulocytes, thrombocytes, leukaemic lymphocytes, and lymphoid line cells.     

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.     

Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli , parasitizing the fat body of cricket Gryllus bimaculatus , by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.     

Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients

Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta-hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios.