Novel Technique For Isolation Research Articles

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Contamination of surfaces for osseointegration of cementless total hip implants by small aluminium oxide particles: analysis of established implants by use of a new technique

BackgroundThe failure of total hip replacements because of wear, particle-induced osteolysis, and aseptic loosening has focussed interest on factors potentially affecting the rate of wear. In this context the effect of particle release from the bone–implant interface of cementless implants is poorly understood. The surface structure for bony ongrowth of many cementless implants is created by grit-blasting. Remnants from this process (Al2O3 particles) on these surfaces have been reported; these remnants have the potential to cause third-body wear. MethodsWe report a novel technique for isolation and quantification of alumina particle contamination. Stems from different manufacturers were electrochemically activated and etched to isolate the alumina residues. After filtration the particles were characterised by scanning electron microscopy and energy-dispersive X-ray analysis. ResultsMany Al2O3 particles were found on all the implants tested. A mean of 426,814 particles per mm2 was measured. Particle size distribution ranged from 0.125 to 66.304μm with a peak in the range 0.25–1μm. ConclusionsOur main finding was a large amount of small Al2O3 particles on all blasted surfaces. On the basis of our results these alumina particle remnants cannot be excluded as a factor causing increased third-body wear.

Um novo modelo de isolamento do tumor de Walker utilizando o gradiente de Ficoll-Hypaque

The purpose was to evaluate a novel technique for isolation of Walker's tumoral cells using a Ficoll-Hypaque gradient and its further influence on tumor development. Twenty male Wistar rats have been divided in 2 groups: G1= without ficoll, G2= with ficoll. Tumor was excised, homogenized and suspended in lactate ringer. A sample of the cell suspension was adjusted at a concentration of 1x10(6) cells/ml (G1). A second sample was centrifuged on a Ficoll-Hypaque gradient and the cell concentration was then adjusted (G2). Tumor was implanted by subcutaneous injection of 1.0 ml in the right armpit of rats. Tumor volume (TV) and tumor weight (TW) were compared in two groups. There were no differences between the two groups in TV (G1=17.9+/-3.8 cm3 vs. G2=17.2+/-4.4 cm3; p=0.190) and TW (G1=7.0+/-1.8 g vs. G2=7.3+/-2.8 g; p=0.569). The histological analysis showed similar patterns of infiltration by small-undifferentiated cells and necrosis in both groups. However, a mild to moderate granulocytic exudate was more frequent in the animals whose tumors derived from Ficoll-isolated cells. Hemorrhage from slight to moderate was only observed in this group. A Ficoll-Hypaque gradient can provide more adequate isolation of Walker's tumor and the cell suspension obtained by this technique has lower contamination by other cell types.

A novel technique for isolation ofFrankiaand generation of single-spore cultures

The conventional isolation techniques for Frankia strains are likely to yield mixed cultures of genetically different strains. This makes genetic studies difficult. We report the development of a method based on calcium alginate beads for the isolation and genetic purification of Frankia strains.Key words: Frankia, alginate beads, isolation, single-spore cultures.

M phase phosphoprotein 10 is a human U3 small nucleolar ribonucleoprotein component.

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

A novel technique for isolation of <i>Frankia</i> and generation of single-spore cultures

A novel technique for isolation of <i>Frankia</i> and generation of single-spore cultures

A Novel Technique for Isolation of Pure Sperm Heads from Disintegrated Mammalian Spermatozoa

The phenomenon of differential charge distribution on sperm surface membrane has been utilised here in a low e.m.f. (electro motive force) capillary electrophoresis system to effect separation of sperm heads from disintegrated mixed spermatozoal subfractions. Washed caudal sperm of goat (Black Bengal variety) and ejaculated washed human sperm were fractionated by sonication into head, mid-piece and tail portions. Routine techniques of density gradient centrifugation on Percoll and/or sucrose were performed with sonicated spermatozoa for separation into their respective subfractions. The products obtained were not free of contamination in either case. Mixed sperm fractions when subjected to the afore mentioned modified capillary electrophoresis technique only the head pieces exhibited high affinity for migration towards the cathode terminal. With this method around 50% of the total sperm heads were separated and collected in absolutely pure form at the cathode side within 2 hrs. at 150 volts (V) and 1.5 milliampere (mA) current at 37.5 degrees C. A 4 cm. long capillary tube with a bore size 1.2 mm. was used for this purpose.

Floating Filters, a Novel Technique for Isolation and Enumeration of Fastidious, Acidophilic, Iron-Oxidizing, Autotrophic Bacteria

Nuclepore polycarbonate filters floating on a liquid, FeSO(4)-containing medium (pH 1.6) were used to isolate a moderately thermophilic bacterium from a pyrite-oxidizing enrichment culture. The isolate failed to grow on any of the conventional solid media tried. To test the general applicability of the method, the enumeration of a fastidious acidophilic organism, Thiobacillus ferrooxidans, was carried out and the results compared with those obtained with other filters, solid media, and the most probable number technique. T. ferrooxidans showed better viability on the floating polycarbonate filters and grew in a much shorter time (4 to 5 days) than with the other techniques.

Novel technique for isolation of human lung mast cells

A novel technique for isolation of human lung mast cells is developed. Human lung tissue was enzymatically digested and the cells were partially purified by centrifugation on Percoll density gradient. Cells obtained at the Percoll density of 1.05–1.09 g/ml were then subjected to a cell sorter equipped with a single argon laser beam (FACS 440). Using four criteria as density, granularity, size and autofluorescence, four major cell populations were identified. One of the major populations contained 70–95% mast cells with a mean and SE values of 88 ± 11% purity, n = 18 as determined by the measurement of total histamine content, light microscopic observation of stained cells with toluidine blue and estrase, and surface-stained IgE fluorescence antibody. Approximately less than 10% mast cells were identified in the three other major cell populations. Mast cells isolated by FACS were found to be intact, viable ( ⋍ 90%) and functionally normal as determined by the release of histamine evoked after stimulation with ionophone A23187, or challenged with anti-human IgE.