Use Of Percoll Research Articles

Estudio de los factores limitantes para la obtención de plantas haploides de Coffea arabica.

El desarrollo de un método eficiente para obtener plantas haploides de café requiere del estudio de los factores más limitantes. En este trabajo se evaluó el desarrollo de los gametos masculinos en relación con el estado fenológico de la flor de C. arabica, se evaluaron tratamientos de desinfección de las flores, tratamientos para el aislamiento y purificación de las microsporas, se estudió el efecto térmico sobre la inducción de la androgénesis y, por último, se evaluaron tratamientos con agentes antimitóticos para la duplicación cromosómica de tejidos haploides. Para inducir el proceso de androgénesis, se determinó que los botones florales entre 1,0 y 1,7 cm de longitud contenían el mayor porcentaje de células gaméticas en los estados uninucleado tardío y binucleado temprano, cruciales para el cambio ontogénico. Se determinó que el menor porcentaje de contaminación microbiana y oxidación biológica se logró con el uso de hipoclorito de calcio combinado con antioxidantes. El aislamiento y purificación de las microsporas se obtuvo mediante maceración mecánica de las anteras y filtración de las células gaméticas en mallas de 40 y 70 micras junto con el empleo de Percoll en gradientes superiores al 50%. La mayor inducción androgénica de las microsporas se consiguió incubando los botones florales desprendidos de la rama a 4°C, observándose divisiones celulares y formación de colonias multicelulares. La aplicación de Colchicina (1%) a meristemos de plantas haploides de C. arabica permitió la obtención de ramas con hojas que duplicaron su material cromosómico y cambiaron su morfología.

Chlorogenic acid improves the quality of boar semen processed in Percoll

This study aimed to evaluate if the addition of chlorogenic acid (ChA) to semen extenders improves the quality of cooled boar semen processed in Percoll. The experimental design was randomized blocks (ejaculates) in a 2×3 factorial (with or without Percoll, and three antioxidant systems: a negative control, without supplementation, a positive control – vitamin E, and ChA), totaling six treatments and 12 repetitions. ChA and vitamin E (VE) were added at 4.5 mg/ml and 400 μg/ml in extender, respectively. At 0, 48 and 72h of storage at 15ºC, 80 ml insemination doses each containing 2.0 billion sperm cells were submitted to centrifugation in Percoll. The use of Percoll impaired (P<0.01) all motility patterns but decreased (P<0.01) the number of abnormal cells at 0, 48 and 72h of storage. Both VE and ChA improved (P<0.05) the total motility after Percoll processing, but only in semen stored for 48h. The same effect was not observed (P>0.05) in semen stored for 72h. ChA improved (P<0.05) the total motility of the semen stored for 72h, but this effect was not observed (P>0.05) when the semen was processed in Percoll. The antioxidants had no effect (P>0.05) on the viability and integrity of the acrosome, but ChA reduced (P<0.05) the number of abnormal cells at 0h, while VE increased the number of abnormal cells in semen stored for 72h, independent of the use of Percoll. There was no effect (P>0.05) of antioxidants or Percoll on the concentration of malondialdehyde in seminal plasma. The use of Percoll had no effect (P>0.05) on the cholesterol efflux, but ChA increased (P<0.05) this parameter at 0h and reduced (P<0.05) in the semen stored for 72h not processed with Percoll. In conclusion, the addition of ChA to semen extenders improved the quality of boar semen processed or not in Percoll.

Conditions for ballisticity and invariance principle for random walk in non-elliptic random environment

We study the asymptotic behaviour of random walks in i.i.d. non-elliptic random environments on $\mathbb{Z} ^d$. Standard conditions for ballisticity and the central limit theorem require ellipticity, and are typically non-local. We use oriented percolation and martingale arguments to find non-trivial local conditions for ballisticity and an annealed invariance principle in the non-elliptic setting. The use of percolation allows certain non-elliptic models to be treated even though ballisticity has not been proved for elliptic perturbations of these models.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine‐stained ischemic brain tissues

We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons.

Separation methods for isolation of human polymorphonuclear leukocytes affect their motile activity

Five commonly used methods for the isolation of human polymorphonuclear leukocytes (PMNLs) from blood and their subsequent effect on cell motile activity were compared. Although all methods (isolation from blood clots, hemolysis of erythrocytes in hypotonic solutions, and sedimentation with the use of Percoll, Ficoll 400 or Dextran T 500 solutions) preserved cell viability, they demonstrated different effects on cell spreading and the speed of spontaneous cell movement. The highest motile activity was shown by PMNLs separated from blood clots and Percoll solutions. In the presence of formylated peptides, N-FMLP-mediated movement was markedly stimulated in PMNLs separated by all five methods, but cells isolated with the use of Dextran T 500 or Ficoll 400 were relatively slower than those isolated with other methods. This suggests that the cells had preserved the sensitivity of their receptors specific for ligands stimulating chemokinesis (chemotaxis) after all five methods of separation. Immunofluorescence observations showed that PMNLs isolated in the presence of FITC-Dextran exhibited polysaccharide-coated surfaces with receptor proteins extended above that coat – an observation which explains why cell coating with polysaccharides does not disturb cell phenotyping with flow cytometry and FACS methods.

Inhibition of oxidative stress and apoptosis enables extended maintenance of integrity and function of isolated hepatocytes in suspension

Isolated hepatocytes in suspension may offer an alternative culture system for bioartificial liver devices. However, maintenance of isolated hepatocyte suspensions in conventional media leads to rapid loss of cell integrity. The aim of this study was to develop a modified medium to better maintain hepatocyte integrity. Isolated rat hepatocytes were prepared by collagenase digestion. Hepatocytes were purified in a Percoll gradient, suspended in bicarbonate buffered isotonic saline supplemented with d-alpha-tocopherol succinate and glucose and medium changed at 24 h (modified medium). The properties of cells treated this way were compared with those prepared by collagenase digestion and suspension in bicarbonate buffered isotonic saline (basic medium). Both media were maintained at 30 degrees C for 48 h on an orbital shaker. Markers for oxidative stress, apoptosis and metabolic function were measured enzymatically. Cell morphology was assessed by electron microscopy. When compared to collagenase-isolated hepatocytes maintained in basic medium, hepatocytes purified by Percoll (Amersham Biosciences, Castle Hill, Australia) and maintained in modified medium demonstrated significantly increased glutathione (GSH) and GSH : glutathione disulphide (GSSH) ratios, decreased lipid peroxidation product formation, decreased caspase-3 protease activity, reduced uptake of trypan blue and loss of lactate dehydrogenase (LDH) and increase preservation of cellular adenosine triphosphate concentration ([ATP]), urea synthesis, ammonia removal and glycogen content. Cell morphology was substantially preserved following 48 h of maintenance in the modified medium. The use of Percoll and modified medium reduces cell injury and apoptosis and greatly improves maintenance of cell function and morphology. The modifications reported here and the use of isolated hepatocyte suspensions in bioartificial liver devices are worthy of further investigation.

Modified flotation method with the use of Percoll for the detection of Isospora suis oocysts in suckling piglet faeces

The modification of flotation method for the examination of diarrhoeic piglet faeces for the detection of Isospora suis oocysts was elaborated. The method was based on removing fractions of fat from the sample of faeces by centrifugation with a 25% Percoll solution. The investigations were carried out in comparison to the McMaster method. From five variants of the Percoll flotation method, the best results were obtained when 2 ml of flotation liquid per 1 g of faeces were used. The limit of detection in the Percoll flotation method was 160 oocysts per 1 g, and was better than with the McMaster method. The efficacy of the modified method was confirmed by results obtained in the examination of the I. suis infected piglets. From all faecal samples, positive samples in the Percoll flotation method were double the results than that of the routine method. Oocysts were first detected by the Percoll flotation method on day 4 post-invasion, i.e. one-day earlier than with the McMaster method. During the experiment (except for 3 days), the extensity of I. suis invasion in the litter examined by the Percoll flotation method was higher than that with the McMaster method. The obtained results show that the modified flotation method with the use of Percoll could be applied in the diagnostics of suckling piglet isosporosis.

A new method to isolate lichen algae by using Percoll®gradient centrifugation

Abstract:A rapid method to isolate intact functional algae from the lichens Evernia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll®gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.

Use of Percoll for the infection of cells in vitro with Cryptosporidium parvum oocysts

A method for the infection of non-adherent THP-1 cells and adherent MDBK cells with Cryptosporidium parvum oocysts using isotonic Percoll solutions was developed. Excystation was maximal after 2 h, but toxicity increased with the oocyst/cell ratio and the incubation time. The infection rates did not increase with the oocyst/cell ratio and both cell types were equally parasitized.

Birth of two infants who were seronegative for human immunodeficiency virus type 1 (HIV-1) after intracytoplasmic injection of sperm from HIV-1-seropositive men

Objective: To report two cases of live births after intracytoplasmic sperm injection (ICSI) in two women who were seronegative for human immunodeficiency virus type 1 (HIV-1) after the use of processed semen from their seropositive husbands. Design: Case reports. Setting: University hospital IVF center. Patient(s): Two HIV-1 seropositive men and their HIV-1 seronegative female partners; all gave their informed consent in writing before undergoing the ICSI procedures. Intervention(s): The men provided semen samples that were processed with the use of Percoll and swim-up techniques. Ovarian stimulation in the women was performed with the long protocol using GnRH analogs and recombinant FSH. ICSI was performed. Main Outcome Measure(s): Oocytes were fertilized by ICSI, and the resulting embryos were transferred to the patients. The mothers and babies were tested for HIV-1 antibodies. Result(s): In the first case, seven mature oocytes were collected and fertilized with ICSI, and three embryos were transferred; the woman became pregnant and gave birth to a healthy boy. Six months after the birth, testing for HIV-1 antibodies in the woman and the baby gave negative results. In the second case, 10 mature oocytes were collected and fertilized with ICSI, and four embryos were transferred; the second woman became pregnant and also gave birth to a healthy boy. Testing for HIV-1 antibodies at the baby’s delivery also gave negative results. Conclusion(s): In women who are infertile because of fallopian tube obstruction or in men who have poor quality semen for artificial insemination, ICSI can be performed using processed semen. This method, which involves the use of only one spermatozoon per oocyte, provides HIV-1 seropositive men with the opportunity to have children with a minimal risk—if any—of infecting their female partners.

Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation

This unit covers the use of Percoll, Nycodenz, and iodixanol for purification of lysosomes from a light mitochondrial pellet or postnuclear supernatant. The first protocol describes isolation of lysosomes from rat liver using a Percoll gradient; it includes some comments on using brain and kidney tissue as source material. Alternatively, this protocol can be modified to enhance the separation of lysosomes by organelle density perturbation. Another alternative uses a discontinuous gradient of Nycodenz to purify lysosomes from a rat liver light mitochondrial pellet. A continuous iodixanol gradient, which can be used to purify other organelles in the light mitochondrial fraction, is yet another alternative for purifying lysosomes. Lysosomes can also be isolated from some of the more commonly used cultured cells. The unit also includes assays for common lysosome marker enzymes.

Sperm Preparation for Intra-uterine Insemination

Controlled ovarian hyperstimulation and sperm injection or intra-uterine insemination (COH/SI) have become important forms of treatment for unexplained and male factor infertility. An extensive literature on sperm preparation has developed (more than 2,000 papers), and the recent withdrawal of indications for human use of Percoll makes it necessary to re-examine the methods available which have been proven effective. In this paper, we have reviewed methods of sperm preparation and their applicability to intra-uterine insemination.

Comparison of two colloidal silica-based sperm separation media with a non–silica-based medium

Objective: To study and compare the effects of three sperm separation media, two silica-based (Percoll; Pharmacia Biotech AB, Uppsala, Sweden, and Isolate; Irvine Scientific, Santa Ana, CA) and one non–silica-based (Ixaprep; Medicult, Copenhagen, Denmark), on the recovery of progressive motile sperm, the percentage of sperm with normal morphology, various sperm motion characteristics determined by computer-aided sperm analysis, and the percentage of acrosome-reacted sperm. Design: Prospective study. Setting: A university-based assisted reproductive technology center. Patient(s): Male partners of couples attending our infertility clinic. Intervention(s): None. Main Outcome Measure(s): Various semen parameters. Result(s): Both Isolate and Ixaprep resulted in enhanced recovery of motile spermatozoa compared with Percoll. The percentage of sperm with forward progressive motility, the percentage of sperm with normal morphology, and various sperm motion characteristics were similar after the use of Percoll and Isolate and were significantly better than after the use of Ixaprep. The same percentage of acrosome-reacted spermatozoa was observed with all three media. Similar results were observed in both normal and subnormal semen samples. Conclusion(s): The use of Isolate and Ixaprep resulted in better recovery of motile spermatozoa. Other semen parameters were similar with the use of Isolate and Percoll, whereas the use of Ixaprep was associated with lower sperm velocities and fewer morphologically normal spermatozoa.

Dynamic aspects concerned with the mechanism of separating motile sperm from nonmotile sperm, leukocytes, and debris with the use of high-density percoll∗ gradients

Objective: To investigate the dynamics and the efficacy of separating motile from nonmotile sperm with the use of Percoll gradients as a function of centrifugation time. Design: In vitro laboratory experiments on human sperm. Setting: Male Infertility Institute. Patient(s): Fresh sperm specimens collected from donors and patients. Intervention(s): Aliquots of sperm specimens, spread on top of 40% and 80% Percoll layers in test tubes, were centrifuged for 5–60 minutes. Main Outcome Measure(s): After centrifugation, samples were aspirated from the bottom, interface, and top layers for microscopic analysis. Result(s): Within 5 minutes, only fast-moving sperm reached the bottom as a result of both centrifugation force and their own tail movements. From 10 minutes onward, nonmotile sperm, slowed down by Percoll, joined them and gradually reversed the separation process. Leukocytes, debris, and abnormal sperm remained in the upper levels and never reached the bottom. Conclusion(s): Separation of motile sperm with the use of Percoll gradients is strongly dependent on centrifugation time. The separation is the gap formed between down-swimming sperm, accelerated by centrifugation, and nonmotile sperm, decelerated by Percoll. This gap gradually disappears with extended spinning.

Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection.

Prior to intracytoplasmic sperm injection (ICSI), seminal fluid is currently washed out from the ejaculated semen and further sperm selection is carried out by a discontinuous Percoll gradient. Possible deleterious effects from the sperm-separating substance Percoll on sperm function or embryo cleavage after in-vitro fertilization (IVF) have, to our knowledge, not yet been reported and the use of Percoll has been widely accepted in IVF. In this study, we examined whether the omission of the Percoll step in the sperm preparation has any influence on the outcome of the ICSI procedure. Two methods of sperm preparation for ICSI were compared in a controlled study on sibling oocytes: washing the semen sample once, followed by a Percoll gradient centrifugation versus washing the sperm sample twice without a Percoll gradient centrifugation. The mean fertilization rates were similar for the two sperm preparation methods: 78.2 +/- 21.4 and 75.0 +/- 24.1% respectively of the intact oocytes displaying two pronuclei. Cleavage rates did not differ statistically between the two groups. Whereas in both groups similar percentages of excellent, good and poor quality embryos were obtained, the percentage of fair quality embryos was significantly higher in the group without Percoll (16.3 +/- 20.1 versus 9.1 +/- 15.7%). However, no statistical differences were observed in either the percentage of transferable embryos or in the percentage of embryos actually transferred or frozen in the two groups. In conclusion, spermatozoa from ejaculates that are washed out from the seminal fluid without any further selection can be used for ICSI without any adverse effect on fertilization and embryo cleavage.

Reexamination of the Intracellular Localization of de Novo Purine Synthesis in Cowpea Nodules.

Sucrose and Percoll density gradient centrifugation were used to separate organelles from the central zone tissue of cowpea (Vigna unguiculata L. Walp. cv Vita 3: Bradyrhizobium strain CB 756) nodules. Enzyme activity analysis has shown that both plastids and mitochondria have a full complement of enzymes for de novo purine synthesis. In vitro activities of individual component enzymes (glycinamide ribonucleotide synthetase, EC 6.3.4.13; glycinamide ribonucleotide transformylase, EC 2.1.2.2; aminoimidazole ribonucleotide synthetase, EC 6.3.3.1; aminoimidazole carboxamide ribonucleotide transformylase, EC 6.3.2.6; and adenylosuccinate-AMP lyase, EC 4.3.2.2) as well as of the whole purine pathway (from ribose-5-phosphate to inosine monophosphate) were similar in the two organelles. No significant cytosolic or bacteroidal activity of any of the purine pathway enzymes was detected on assay. These findings are contrary to earlier studies (M.J. Boland, K.R. Schubert [1983] Arch Biochem Biophys 220: 179-187; B.J. Shelp C.A. Atkins, P.J. Storer, D.T. Canvin [1983] Arch Biochem Biophys 224: 429-441) that concluded that enhanced expression of purine synthesis in nodules of ureide-forming species is localized to plastids. Significantly increased recovery of activity of key pathway enzymes (particularly of labile aminoimidazole ribonucleotide synthetase) coupled with improved assay methods and the use of Percoll in addition to sucrose for gradient centrifugation have together contributed to much higher reaction rates and more definitive analyses of particulate fractions.

Amenability, Kazhdan’s property and percolation for trees, groups and equivalence relations

We prove amenability for a broad class of equivalence relations which have trees associated to the equivalence classes. The proof makes crucial use of percolation on trees. We also discuss related concepts and results, including amenability of automorphism groups. A second main result is that no discrete subgroup of the automorphism group of a tree is isomorphic to the fundamental group of any closed manifoldM admitting a nontrivial connection-preserving, volume-preserving action of a noncompact, simply connected, almost simple Lie group having Kazhdan’s property (T). The technique of proof also shows that M does not admit a hyperbolic structure.

Are human spermatozoa separated on a Percoll density gradient safe for therapeutic use?

Motile morphologically normal human spermatozoa can be separated from semen by buoyant density centrifugation on Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden) gradients. In this study, the authors have examined (1) the efficiency of washing procedures to remove contaminating Percoll particles from the separated spermatozoa, and (2) the potential of Percoll particles, which contain silica, to cause an inflammatory response when used for intrauterine insemination, or when introduced into the fallopian tube during gamete intrafallopian transfer (GIFT) procedures, as assessed by an intraperitoneal injection into mice. Although Percoll was phagocytosed at the injection site, and therefore cannot be presumed to be totally inert, no generalized inflammatory response was detected. A double spin and wash technique was found to remove most residual Percoll from the spermatozoa, as assessed by scanning electron microscopy. These results suggest that procedures involving the use of Percoll for the separation of human spermatozoa for in vitro fertilization, GIFT, or intrauterine insemination should include stringent washing protocols that will remove most, if not all, contaminating Percoll from the sample.

On the use of Percoll as a marker for liposomes in electron microscopy of mouse spleen tissue.

Liposomes encapsulating Percoll were prepared by reverse phase evaporation and characterized by electron microscopy (EM). Encapsulated Percoll was contained between lipid bilayers and in the lumen of the liposomes. The largest population of liposomes had diameters between 110 and 140 nm (22.6 per cent of total vesicle population). Percoll proved to be a good marker for liposomes in spleen tissue. It is inherently electron dense, has dimensions which are uniform and not easily confused with subcellular organelles, and can be encapsulated and visualized by EM examination of tissue by routine procedures. Electron microscope examination of spleen tissue excised 20 min after intravenous injection of liposomes encapsulating Percoll showed the presence of both liposome-encapsulated Percoll and free Percoll in phagolysosomes, suggesting that liposomes had been phagocytosed by macrophages in a way similar to circulating blood cells and that enzymatic digestion had already begun. On the other hand, intravenously injected free Percoll did not localize in phagolysosomes, but its fate appeared similar to that of intravenously injected colloidal carbon, being phagocytosed by what appeared to be macrophages and platelets.