Suitable procedures for separation of various stages of Toxoplasma gondii by density gradient centrifugation have been delineated using peritoneal exudate of infected mouse as a model. Separation from host cells was effected by gradient centrifugation at 450 g for 30 minutes. Using Ficoll, dextran and sucrose, average parasite recoveries by pooling up to and including the peak fraction of 73.65%, 66.18% and 65.68% respectively were obtained. Toxoplasma trophozoites peaked at density of 1.040 g/ml with Ficoll, 1.060 g/ml with dextran and 1.110 g/ml with sucrose. In view of successful separation of exo-enteric stages of Toxoplasma by density gradient centrifugation, possible application of this method to isolation of various endo-enteric stages is discussed.

• MeSH
• centrifugace - gradient hustoty * MeSH
• dextrany MeSH
• Ficoll MeSH
• myši MeSH
• Toxoplasma cytologie   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dextrany MeSH
• Ficoll MeSH

In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.

• MeSH
• centrifugace - gradient hustoty metody   MeSH
• kryoprezervace metody   MeSH
• motilita spermií fyziologie   MeSH
• oxid křemičitý chemie   MeSH
• povidon chemie   MeSH
• proteomika MeSH
• ryby fyziologie   MeSH
• spermie fyziologie   MeSH
• uchování spermatu metody   MeSH
• viabilita buněk fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

• MeSH
• analýza spermatu MeSH
• centrifugace - gradient hustoty veterinární   MeSH
• kryoprezervace veterinární   MeSH
• separace buněk metody   veterinární   MeSH
• uchování spermatu veterinární   MeSH
• vysoká zvěř fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.

• MeSH
• Arabidopsis cytologie   MeSH
• časové faktory MeSH
• centrifugace - gradient hustoty ekonomika   metody   MeSH
• oxid křemičitý chemie   MeSH
• povidon chemie   MeSH
• pyl cytologie   MeSH
• separace buněk ekonomika   metody   MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

Mitochondria isolated from Neurospora crassa were purified by centrifugation in a Percoll density gradient. Enzyme activities and cytochrome differential spectra revealed a high purity of the mitochondria. As compared with a crude mitochondrial fraction the purified mitochondria exhibited a high respiratory activity and a fine ADP/O ratio. Electrophoresis of nucleic acids demonstrated the absence of cytoplasmic rRNA.

• MeSH
• adenosindifosfát analýza   MeSH
• centrifugace - gradient hustoty MeSH
• cytochromy analýza   MeSH
• fungální RNA analýza   MeSH
• L-laktátdehydrogenasa analýza   MeSH
• mitochondrie analýza   enzymologie   MeSH
• Neurospora crassa ultrastruktura   MeSH
• Neurospora ultrastruktura   MeSH
• respirační komplex IV analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosindifosfát MeSH
• cytochromy MeSH
• fungální RNA MeSH
• L-laktátdehydrogenasa MeSH
• respirační komplex IV MeSH

An attempt was made to assess whether the choice of the gradient media could influence the yield of basolateral membrane vesicles isolated from the rat intestine as well as their functional characteristics. Crude membranes prepared in the same way were therefore centrifuged with 10% Percoll, on a discontinuous sucrose gradient or on a continuous sorbitol gradient. The protein yield was significantly higher with the Percoll gradient than with sucrose and sorbitol gradient centrifugation (2.7 +/- 1.0%; 0.4 +/- 0.1%; 0.6 +/- 0.2%, respectively). Enrichment in Na+,K+-ATPase was similar in all three preparations (8.50 +/- 2.34; 8.22 +/- 4.78; 8.20 +/- 2.08). However, contamination with brush border membranes was significantly higher after Percoll gradient centrifugation and negligible after the use of the other two gradient media. Transport of D-glucose in the BLM prepared by Percoll gradient centrifugation also indicated some contamination with functional brush-border membranes. An attempt to purify basolateral membrane vesicles after Percoll gradient centrifugation with Ca2+ precipitation, however, reduced the protein yield to less than 1%. We conclude that in the preparation of basolateral membrane vesicles from the rat enterocytes each of the gradient media may have certain advantages and disadvantages, which should be considered according to the purpose of the preparation.

• MeSH
• buněčná membrána * MeSH
• centrifugace - gradient hustoty metody   MeSH
• frakcionace buněk metody   MeSH
• glukosa MeSH
• krysa rodu Rattus MeSH
• oxid křemičitý MeSH
• potkani inbrední WKY MeSH
• povidon MeSH
• sacharosa MeSH
• sorbitol MeSH
• střeva cytologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH
• sacharosa MeSH
• sorbitol MeSH

PURPOSE: To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse. METHODS: Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro. RESULTS: Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer. CONCLUSIONS: These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12(-)p63(+) population being closer to the primitive limbal SC.

• MeSH
• ABC transportér z rodiny G, člen 2 MeSH
• ABC transportéry analýza   genetika   MeSH
• buněčné dělení MeSH
• buňky 3T3 MeSH
• centrifugace - gradient hustoty metody   MeSH
• fibroblasty cytologie   MeSH
• kmenové buňky cytologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• oxid křemičitý MeSH
• polymerázová řetězová reakce MeSH
• povidon MeSH
• průtoková cytometrie MeSH
• rohovkový epitel cytologie   MeSH
• separace buněk metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportér z rodiny G, člen 2 MeSH
• ABC transportéry MeSH
• Abcg2 protein, mouse MeSH Prohlížeč
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

We attempted to select a fraction of common carp, Cyprinus carpio spermatozoa that best survived a conventional freeze/thaw procedure, by centrifugation of frozen/thawed sperm through a Percoll gradient (45% and 90%). The proportion of motile spermatozoa (65.81 ± 5.19%), their velocity (77.58 ± 31.07 μm/sec), and membrane integrity (83.66 ± 4.38% intact) were significantly higher in separated sperm than in whole samples (motility 23.36 ± 2.98%, velocity 55.55 ± 19.03 μm/sec, and membrane integrity 57.92 ± 4.65%). Our results demonstrated that Percoll gradient centrifugation shows promise as a technique for selecting high quality cryopreserved fish spermatozoa, which could be useful for cryobiological research. Further studies are needed to evaluate the potentially higher fertilizing ability of the separated spermatozoa.

• MeSH
• buněčná membrána fyziologie   ultrastruktura   MeSH
• centrifugace - gradient hustoty veterinární   MeSH
• kapři * MeSH
• kryoprezervace veterinární   MeSH
• motilita spermií * MeSH
• oxid křemičitý chemie   MeSH
• povidon chemie   MeSH
• spermie fyziologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

A mixture of Ficoll 400 and sodium diatrizoate (Hypaque) at a density of 1.077 g/ml has been used to isolate the mononuclear cells from the remaining haematic cells. A simple, inexpensive and classical method was established to obtain substantially erythrocyte-free polymorphonuclear cell preparations from mouse peripheral blood, using a mixture of the same substances but at a density of 1.119 g/ml. This method along with that at a density of 1.077 g/ml allows two cellular bands to appear which contain mononuclear and polymorphonuclear (PMN) cells, respectively. Using this method, the counts of monocytes isolated from peripheral blood are significantly greater than those obtained by a one-step Ficoll-Hypaque procedure. On the contrary, the counts of PMN cells are significantly smaller than when sedimentation in dextran (6% solution) is used after gradient centrifugation. In this paper, chemiluminescence assay has been used to analyze the possible variations in phagocytic activity of cells isolated by both procedures, since it appears to be one of the most sensitive methods available for this purpose. The results obtained show a slightly greater activation in monocytes and PMN cells isolated by one-step Ficoll-Hypaque procedure, in comparison with another method which uses both Ficoll-Hypaque 1077 and Ficoll-Hypaque 1119, although statistical differences were not significant.

• MeSH
• centrifugace - gradient hustoty MeSH
• diatrizoát MeSH
• fagocytóza MeSH
• fagocyty cytologie   fyziologie   MeSH
• Ficoll MeSH
• luminiscenční měření MeSH
• monocyty cytologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• neutrofily cytologie   fyziologie   MeSH
• separace buněk metody   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• diatrizoát MeSH
• Ficoll MeSH

More than one 80S monosome can translate an mRNA molecule at a time producing polysomes. The most widely used method to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes is a high-velocity centrifugation of whole cell extracts in linear sucrose gradients. This polysome profile analysis technique has been routinely used to monitor translational fitness of cells under a variety of physiological conditions, to investigate functions of initiation factors involved in translation, to reveal defects in ribosome biogenesis, to determine roles of 5' UTR structures on mRNA translatability, and more recently for examination of miRNA-mediated translational repression (see an application of this protocol on Polysome analysis for determining mRNA and ribosome association in Saccharomyces cerevisiae).

• Klíčová slova
• Grow yeast cultures, Polysome profile analysis, Sucrose density gradient centrifugation, Western and Northern blotting, Yeast whole cell extracts (WCEs),
• MeSH
• centrifugace - gradient hustoty metody   MeSH
• northern blotting metody   MeSH
• polyribozomy chemie   genetika   MeSH
• Saccharomyces cerevisiae chemie   cytologie   genetika   MeSH
• sacharosa chemie   MeSH
• western blotting metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sacharosa MeSH