On top of the sand bed sits a supernatant layer of unpurified water.
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Phage proteins can be detected in the supernatant about 10 minutes after infection (37°).
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Supernatant was removed and periodide crystals dissolved in 900 μl of 1,2-dichloroethane.
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The cleaved DNA fragments are then liberated into the supernatant by incubating the nuclei for an hour before the nuclei is pelleted by centrifugation. The DNA fragments are then extracted from the supernatant and can be used to construct a sequencing library.
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The genomic material remaining in the supernatant is extracted and physically separated from the mRNA.
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During the fourth stage the outlet valve opens and the "clean" supernatant liquor exits the tank.
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Plumbic acetate gives a purplish-red precipitate, mercuric cyanide a blue one, the supernatant liquid being also blue.
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Fecal sludge from public toilets took longer to dewater than sludge from other sources, and had turbid supernatant after settling.
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The extract was left standing again overnight, and the remaining supernatant was obtained through centrifugation. The dissolved proteins were precipitated by saturation of the supernatant using ammonium sulfate. The proteins were obtained through centrifugation, redissolving in H20 and dialysis against running water for two days. Finally, the precipitate was removed via low speed centrifugation and the supernatant was freeze-dried.S. Olsnes, T. Haylett en K. Refsnes, „Purification and Characterisation of the highly toxic Lectin Modeccin,” The Journal of Biological Chemistry, vol. 253, nr. 14, pp. 5069-5073, 1978.
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Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant can yield 1 to 60 μg/ml of monoclonal antibody, which is maintained at -20 °C or lower until required. By using culture supernatant or a purified immunoglobulin preparation, further analysis of a potential monoclonal antibody producing hybridoma can be made in terms of reactivity, specificity, and cross-reactivity.
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Macrophages also play a role in inducing the proliferation of Schwann cells that occurs during Wallerian degeneration. Supernatant has been collected from medium in which macrophages are active in myelin phagocytosis where lysosomal processing of the myelin occurs within the macrophage. The supernatant contains a mitogenic factor, a mitosis promoting factor, that is characterized heat and trypsin sensitivity, both of which characterize it as a peptide. Treatment of Schwann cells with the collected supernatant shows that it is a mitogenic factor and thus plays an important role in the proliferation of Schwann cells.
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The blood is mixed with a small amount of sterile water to cause hemolysis of the RBCs, yielding free hemoglobin. The sample is next centrifuged for several minutes. The pink hemoglobin-containing supernatant is then mixed with 1 mL of 1% NaOH for each 5 mL of supernatant. The color of the fluid is assessed after 2 minutes.
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OPN has also been reported to be increased in the sputum supernatant of smoking asthmatics, as well as the BALF and bronchial tissue of smoking controls and asthmatics.
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Plasma is then processed again by centrifugation to remove residual intact blood cells. The supernatant is used for DNA extraction, which can be performed using commercially available kits.
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When the diluent is known (as is the case for a chemical formulation), additional diluent can be prepared. If the diluent is unknown, equilibrium supernatant is readily obtained by centrifugation.
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To obtain serum, a blood sample is allowed to clot (coagulation). The sample is then centrifuged to remove the clot and blood cells, and the resulting liquid supernatant is serum.
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During culture, supernatant (containing idiotype) is collected until sufficient amounts have been produced to yield adequate dosage of vaccine. This supernatant is purified by affinity chromatography and conjugated (bonded) to keyhole limpet haemocyanin (KLH), an immune-stimulating carrier protein, resulting in a finished vaccine that can be shipped and administered to patients. In the Phase III clinical trial, manufacturing success was approximately 95% of treated patients. The BiovaxID vaccine is manufactured through a process known as rescue fusion hybridization.
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The mixture is separated into eluate (m6A+ RNAs) and supernatant (m6A- RNAs) pools. External RNA Controls Consortium (ERCC) spike ins are added to the eluate and supernatant, as well as an independent control arm consisting of just ERCC spike in. After antibody cleavage in the eluate pool, each of the three mixtures are sequenced on a next generation sequencing platform. The m6A levels per site or gene could be quantified by the ERCC-normalized RNA abundances in different pools.
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In 1980, it was discovered that B. pertussis could attach to hamster tracheal epithelial (HTE) cells, and also, that the supernatant from the cultured bacterium could disrupt the cell cycle of uninfected cells. This prompted the scientists W. E. Goldman, D. G. Klapper, and J. B. Baseman to isolate and characterize a novel substance from B. pertussis supernatant. The novel disaccharide tetrapeptide that they had purified showed toxicity for HTE cells and tracheal ring cultures. Subsequently, they named the newly sequestered molecule tracheal cytotoxin (TCT).
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After centrifugation the supernatant solution is removed, leaving a pellet of crude DNA. Whether the pellet is visible depends on the amount of DNA and on its purity (dirtier pellets are easier to see) or the use of co-precipitants. In the next step, 70% ethanol is added to the pellet, and it is gently mixed to break the pellet loose and wash it. This removes some of the salts present in the leftover supernatant and bound to DNA pellet making the final DNA cleaner.
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Cancer cell line supernatant is an attractive source of secreted proteins. There are many standardized cell lines available and supernatant is much simpler to analyze than proximal body fluid. But it is unclear whether a cell line secretome is a good representation of an actual tumor in its specific microenvironment and a standardized cell line is not illustrative of the heterogeneity of a real tumor. Analysis of proximal fluids can give a better idea of a human tumor secretome, but this method also has its drawbacks.
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The enrichment of amplified beads is achieved through hybridization to a larger, low density, non-magnetic polystyrene beads that pre-loaded with a biotinylated capture oligonucleotides (DNA sequence that complementary to ePCR amplicon sequence). The mixture is then centrifuged to separate the amplified and capture beads complex from the unamplified beads. The amplified, capture bead complex has a lower density and thus will remain in the supernatant while the unamplified beads form a pellet. The supernatant is recovered and treated with NaOH which will break the complex.
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After 5 minutes, carefully decant the supernatant solution and determine the fluoride. Calculate the difference between the original and treated water fluoride concentration. Multiply the difference by 100 to give the fluoride uptake capacity of AA in mg/kg.
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For isolation of slotoxin, scorpions of the species Centruroides noxius are milked for venom in the laboratory. The crude venom is being dissolved in distilled water and spun. The supernatant is separated. The active fraction is then further separated.
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ISG15 is secreted from the cell and can be detected in supernatant or blood plasma. ISG15 binds the LFA-1 integrin receptor on NK- and T-cells to potentiate their production of IFN-II, which is essential for mycobacterial immunity.
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This design allows the solid cake and the liquid centrate to leave the centrifuge bowl at opposite ends. For this design, the conveyor pushes the sludge towards the end streams and the supernatant liquid is allowed to exit over the weirs.
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A 52±4 kD molecular weight protein, isolatable from a supernatant of the cell line M20-2, said protein being characterized by a pI of 4.1 ± 0.2 and an ability to inhibit or reduce an IL-1 mediated inflammatory response.
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Binding of mouse immunoglobulins is restricted to those having VκI light chains. Given these specific requirements for effective binding, the main application for immobilized Protein L is purification of monoclonal antibodies from ascites or cell culture supernatant that are known to have the kappa light chain. Protein L is extremely useful for purification of VLκ-containing monoclonal antibodies from culture supernatant because it does not bind bovine immunoglobulins, which are often present in the media as a serum supplement. Also, Protein L does not interfere with the antigen-binding site of the antibody, making it useful for immunoprecipitation assays, even using IgM.
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Thus, the differential centrifugation method is the successive pelleting of particles from the previous supernatant, using increasingly higher centrifugation forces. Cellular organelles separated by differential centrifugation maintain a relatively high degree of normal functioning, as long as they are not subject to denaturing conditions during isolation.
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Continue the incubation to allow antibody-antigen complexes to form. # Precipitate the complex of interest, removing it from bulk solution. # Wash precipitated complex several times. Spin each time between washes when using agarose beads or place tube on magnet when using superparamagnetic beads and then remove the supernatant.
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When suspensions of particles are "spun" in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid. Non-compacted particles remain mostly in the liquid called "supernatant" and can be removed from the vessel thereby separating the supernatant from the pellet. The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force.
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Culture techniques and media vary depending upon the requirements of the fungal isolate involved, however the general procedure consist of the following: fungal hyphae are typically placed in liquid growth media and placed in shake culture until the fungal culture has increased in biomass. The fungal hyphae are removed from the growth media, washed with distilled water to remove the growth media, placed in distilled water and incubated on shake culture for 24 to 48 hours. The fungal hyphae are separated from the supernatant, and an aliquot of the supernatant is added to 1.0 mM ion solution. The ion solution is then monitored for 2 to 3 days for the formation of nanoparticles.
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After the final wash, remove as much supernatant as possible. # Elute proteins from the solid support using low-pH or SDS sample loading buffer. # Analyze complexes or antigens of interest. This can be done in a variety of ways: ## SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) followed by gel staining.
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Detection of viral hemagglutinin also has been recommended as a diagnostic technique. Tissues are triturated in diluent and then sedimented by centrifugation. The supernatant fluid is tested for agglutinating activity for guinea pig erythrocytes. This test requires a minimum of laboratory equipment and is effective in the absence of antibody.
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The precipitated protein (curd) is separated from the supernatant (whey) by filtration or centrifugation. The curd must be washed in order to remove residues of whey solubles. Subsequently, the pH is neutralised and readjusted to 7, and a dry protein isolate is obtained with a final mechanical drying step, called spray- drying.
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In order to purify toxin B from C. difficile cell cultures, brain heart infusion broth is used because it promotes the synthesis of toxin B. The filtration method facilitates purification of toxin B from the supernatant of C. difficile. The toxin concentration of the supernatant is proportional to the organism cell count. It has been proposed by many studies that the majority of the toxins are released in either late log phase or early stationary phases, hence, toxin B is continuously secreted by cells. Although there are many methods employed by different studies in purifying toxin B, the majority of studies use methods involving concentrations of ultrafiltrated ammonium sulfate or precipitation, in lieu by either gel filtration or ion-exchange chromatography.
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Mixed sludge and sewage is decanted at the surface and separated into supernatant and sludge components. The efficiency of deep shaft treatment can be high. Surface aerators are commonly quoted as having an aeration efficiency of 0.5 - 1.5 kg O2/kWh, diffused aeration as 1.5 - 2.5 kg O2/KWh. Deep Shaft claims 5 – 8 kg O2/kWh.
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An amniotic fluid sample is collected via amniocentesis and the sample is spun down in a centrifuge at 1000 rpm for 3–5 minutes. Thin layer chromatography (TLC) is performed on the supernatant, which separates out the components. Lecithin and sphingomyelin are relatively easy to identify on TLC and the predictive value of the test is good.
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A) Electron micrograph of poxvirus particles in synovium of a big brown bat, northwestern United States. B) Negative staining of poxvirus particles in cell culture supernatant. Scale bar = 100 nm. Poxviridae viral particles (virions) are generally enveloped (external enveloped virion), though the intracellular mature virion form of the virus, which contains different envelope, is also infectious.
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The supernatant water is then run back into the treatment process or disposed of as a waste-water stream. In some countries, the sludge may be used as a soil conditioner. Inadequate filter maintenance has been the cause of occasional drinking water contamination. Sand filters are occasionally used in the sewage treatment as a final polishing stage.
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The solution is then centrifuged twice, first for 10 minutes to remove any unexfoliated black phosphorus and then for 20 minutes at a higher speed to separate thick layers of phosphorene (5–12 layers) from NMP. The supernatant then is centrifuged again at higher speed for another 20 minutes to separate thinner layers of phosphorene (1–7 layers).
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Rock sample preparation for illite crystallinity can vary slightly, but boils down to basically the same steps. Although for accurate returns in testing, consistency of sample preparation is a must. General sample preparation for illite crystallinity is as follows: # Rinse and dry the field sample # Crush the sample # Stir sample into deionized water and let settle overnight to isolate clay sized particles (<2 μm) # Dry supernatant containing <2 μm particles # Mix with deionized water and centrifuge # Collect supernatant containing <2 μm particles # Centrifuge <2 μm particle solution and dry # Mix with water and deposit on a glass slide The sample is first broken down, using the steps above, and prepared for XRD analysis. Results from the XRD are then compared to pre-established values assigned to metamorphic zones/metamorphic facies.
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Impurities remain in the supernatant liquid. In some cases crystals do not form quickly and the solution remains supersaturated after cooling. This is because there is a thermodynamic barrier to the formation of a crystal in a liquid medium. Commonly this is overcome by adding a tiny crystal of the solute compound to the supersaturated solution, a process known as "seeding".
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This suspension is centrifuged again to once again pellet DNA and the supernatant solution is removed. This step is repeated once. Finally, the pellet is air- dried and the DNA is resuspended in water or other desired buffer. It is important not to over-dry the pellet as it may lead to denaturation of DNA and make it harder to resuspend.
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The superheated limed juice is then allowed to flash to its saturation temperature: this process precipitates impurities which get held up in the calcium carbonate crystals. The flashed juice is then transferred to a clarification tank which allows the suspended solids to settle. The supernatant, known as clear juice is drawn off of the clarifier and sent to the evaporators.
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The classical immunoprecipitation technique is then applied: magnetic beads conjugated to anti-mouse-IgG are used to bind the anti-5mC antibodies, and unbound DNA is removed in the supernatant. To purify the DNA, proteinase K is added to digest the antibodies and release the DNA, which can be collected and prepared for DNA detection. For more details regarding the experimental steps see.
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It is also important to block the thiol functions to avoid air oxidation and the loss of proteolytic activity. To eliminate organic and insoluble molecules, the sample is first filtered and afterwards centrifuged at 11000g for 30min. The pellet is discarded and the supernatant added to 96% alcohol with a ratio of 1:3. Impurities precipitate and can be eliminated by filtration.
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Orange juice is a suspension that consists of heterogeneous particles in a clear serum. A serum is the clear supernatant after the precipitation of the cloud through centrifugation. The previously mentioned cloud makes up a large part of the suspension. If the suspension in orange juice is not stable, the cloud particles can flocculate which causes the suspension to physically decompose.
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Edible soy protein "isolate" is derived from defatted soy flour with a high solubility in water (high NSI). The aqueous extraction is carried out at a pH below 9. The extract is clarified to remove the insoluble material and the supernatant liquid is acidified to a pH range of 4-5. The precipitated protein-curd is collected and separated from the whey by centrifuge.
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Weissella is a genus of gram-positive bacteria, placed within the family Leuconostocaceae, and formerly considered species of the Leuconostoc paramesenteroides group. The morphology of Weissella species varies from spherical or lenticular cells to irregular rods. Several strains of Weissella cibaria and Weissella confusa have shown the probiotic potential . In particular, the cell-free culture supernatant of Weissella confusa shows various beneficial characteristics such as antibacterial potential and anti- inflammatory efficiency .
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All these measuring techniques may require dilution of the sample. Sometimes this dilution might affect properties of the sample and change zeta potential. There is only one justified way to perform this dilution – by using equilibrium supernatant. In this case, the interfacial equilibrium between the surface and the bulk liquid would be maintained and zeta potential would be the same for all volume fractions of particles in the suspension.
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The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. The first screening technique used is called ELISA. The hybridoma culture supernatant, secondary enzyme labeled conjugate, and chromogenic substrate, are then incubated, and the formation of a colored product indicates a positive hybridoma. Alternatively, immunocytochemical, western blot, and immunoprecipitation-mass spectrometry screening can also be used.
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Afterwards, (NH4)2SO4 fractioning is done by addition of this substance at a concentration of 0.472 mg/ml. Chymopapain precipitates and can be retrieved through another centrifugation, again at 11000g for 30min. The supernatant is discarded and the ion exchange chromatography can be carried out, with a linear gradient of 100mM (Na+) and different volumes of elution. Studying A280 chymopapain is found in the fraction of 750-1000 ml.
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The waters of the upper two pools and the waterfall may be too hot for bathing. Supernatant liquid is a layer of light pure cool water that can float on top of the denser, hot, mineral-rich hot springs outflow. Don't test by dipping toes or fingers into the upper pools. The hot fluids below may be hot enough to scald and make second and third degree burns.
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They can easily be transfected either with viral supernatant or physical methods. Even mRNA can be shuttled into the cells with high efficiency. Since no integration of mRNA into the genome occurs, this transfection is less risky. NK-92 cells have also been transfected with a high affinity Fc Receptor (NK-92Fc) which is the main receptor for monoclonal antibodies to execute antibody dependent cellular cytotoxicty (ADCC), including rituximab and ofatumumab.
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Produced mainly in parts of the world where pineapples are grown, such as Thailand or Malaysia, bromelain is extracted from the peel, stem, leaves or waste of the pineapple plant after processing the fruit for juice or other purposes. The starting material is blended and pressed through a filter to obtain a supernatant liquid containing the soluble bromelain enzyme. Further processing includes purification and concentration of the enzyme.
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The 1992 Indo-Tibetan Border Police Expedition to Mount Everest by Indo-Tibetan Border Police,led by Additional Deputy Supernatant Major Hukam Singh on 1992 recorded a total of 8 ascents by Indians including Ms.Santosh Yadav. The second woman summitter from India. Suresh Kumar was member in the expedition as the film team . Indo-Tibetan Border Police, of which senior medical officer Chittaranjan R. Pattanayak was the deputy leader.
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After finishing the digestion the peptides generated in this process have to be extracted from the gel matrix. This is accomplished by one or several extraction steps. The gel particles are incubated with an extraction solution and the supernatant is collected. In the first extraction, almost all of the peptide is recovered, the repetition of the extraction step can increase the yield of the whole process by only 5-10%.
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The supernatant containing contaminants can be carefully removed so as not to disturb the beads. The wash buffer can then be added to the beads and after mixing, the beads are again separated by centrifugation. With superparamagnetic beads, the sample is placed in a magnetic field so that the beads can collect on the side of the tube. This procedure is generally complete in approximately 30 seconds, and the remaining (unwanted) liquid is pipetted away.
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Mitotic selection is a drug- free procedure for the selection of mitotic cells from a monolayer undergoing exponential growth. During mitosis, cells undergo changes in morphology, and mitotic selection takes advantage of this in adherent cells grown in a monolayer. The cells become more spherical, decreasing the surface area of cell membrane attached to the culture plate. Mitotic cells can therefore be completely detached by gently shaking and collected from the supernatant.
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Differential centrifugation, on the other hand, does not utilize a density gradient, and the centrifugation is taken in increasingly speeds. The different centrifugation speeds often create separation into not more than two fractions, so the supernatant can be separated further in additional centrifugation steps. For that, each step the centrifugation speed has to be increased until the desired particles are separated. In contrast, the density gradient centrifugation is usually performed with just one centrifugation speed.
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There are some differences between hPL manufacturing protocols, but they all share the same core of being frozen at very low temperatures and thawed. This process may be repeated two or three times to cause complete platelet lysis. The resultant hPL can then undergo different manufacturing steps to achieve multiple grades of hPL. The most common form of hPL undergoes few processing steps, producing a product made of the supernatant following the freeze/thaw process.
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The supernatant liquid (containing many of the fission products) was separated from the solid. The precipitate was then dissolved in nitric acid before the addition of an oxidant (such as potassium permanganate) to produce PuO22+. The plutonium was maintained in the +6 oxidation state by addition of a dichromate salt. The bismuth phosphate was next re-precipitated, leaving the plutonium in solution, and an iron(II) salt (such as ferrous sulfate) was added.
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Then the supernatant in each well can be checked for the desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as monoclonal antibodies. The production of monoclonal antibodies was first invented by César Milstein and Georges J. F. Köhler, which earned them the 1984 Nobel Prize in Physiology or Medicine, shared with Niels Kaj Jerne.
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UcfDNA is classified into two categories based on its size: high-molecular-weight and low-molecular-weight. High-molecular- weight ucfDNA refers to DNA fragments in urine with the size longer than a kilobase pair (kbp). They are separated together with cell debris by centrifugation of urine and they represent the genome of the originated cells. In contrast, low-molecular-weight ucfDNA is DNA fragments in urine sized with hundreds of bp, and appears in the supernatant after centrifugation.
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The archaeocins produced by Sulfolobus are entirely different from halocins, since their activity is predominantly associated with the cells and not the supernatant. To date, the spectrum of sulfolobicin activity appears to be restricted to other members of the Sulfolobales: the sulfolobicin inhibited S. solfataricus P1, S. shibatae B12, and six nonproducing strains of S. islandicus. Activity appears to be archaeocidal but not archaeolytic. Two genes involved in sulfolobicin production have been identified in S. acidocaldarius and S. tokodaii.
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In 1956, Arthur Kornberg and his colleagues discovered Pol I by using Escherichia coli (E. coli) extracts to develop a DNA synthesis assay. The scientists added 14C-labeled thymidine so that a radioactive polymer of DNA, not RNA, could be retrieved. To initiate the purification of DNA polymerase, the scientists added streptomycin sulfate to the E. coli extract which created a precipitate that consisted of nucleic acid-free supernatant (S-fraction) and nucleic acid-containing precipitate (P-fraction).
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Cellular thermal shift assay (CETSA) is a biophysical technique applicable on living cells as well as tissue biopsies. CETSA is based on the discovery that protein melting curves can also be generated in intact cells and that drug binding leads to very significant thermal stabilization of proteins. Upon denaturation, proteins are aggregated and can thus be removed by centrifugation after lysis of the cells. The stable proteins are found in the supernatant can be detected; e.g.
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TcdA and TcdB are present in supernatant fluids of Clostridium difficile cultures and can be purified from filtrates. Both toxins are consistently detected in fecal samples from humans and animals and are now used as markers to diagnose C. difficile infection. Over 90% of patients infected with C. difficile were found to have cytotoxic activity in their stool. Glucosylation of Rho GTPases inactivates the GTPase proteins, leading to collapse of the cytoskelton, resulting in cell rounding.
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Structurally, phenanthriplatin is similar to cisplatin, differing only in the presence of a phenanthridine ligand instead of a chloride in its structure. To synthesise phenanthriplatin, a one equivalent of silver nitrate is added to solution of cisplatin in dimethylformamide. The mixture is stirred at 55 °C away from light and the resulting silver chloride precipitate is filtered out. Next, phenanthridine is added to the supernatant and this is also mixed at 55 °C for 16 hours.
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In Tamil Nadu, a plain rice porridge, or the thick supernatant water from overcooked rice, is known as (). Kanji is also prepared with different grains available in different parts of Tamil Nadu, for example minor millet or pearl millet, finger millet, broken wheat, maize. The people of Kerala also call this preparation of rice in a watery state , and it is eaten as a porridge with green lentils or chutney. is prepared with rice or ragi.
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This is known as zone settling, because it is easy to make a distinction between several different zones which separated by concentration discontinuities. Fig. 3 represents a typical batch-settling column tests on a suspension exhibiting zone-settling characteristics. There is a clear interface near the top of the column would be formed to separating the settling sludge mass from the clarified supernatant as long as leaving such a suspension to stand in a settling column.
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Antibodies from Lymphocyte Secretion or Antibody in Lymphocyte Supernatant or ALS Assay is an immunological assay to detect active diseases like tuberculosis, cholera, typhoid etc. Recently, ALS assay nods the scientific community as it is rapidly used for diagnosis of tuberculosis. The principle is based on the secretion of antibody from in vivo activated plasma B cells found in blood circulation for a short period of time in response to TB-antigens during active TB infection rather than latent TB infection.
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Chromatographic processes began to take shape in 1983. In the 1990s, the Zenalb and the CSL Albumex processes were created which incorporated chromatography with a few variations. The general approach to using chromatography for plasma fractionation for albumin is: recovery of supernatant I, delipidation, anion exchange chromatography, cation exchange chromatography, and gel filtration chromatography. The recovered purified material is formulated with combinations of sodium octanoate and sodium N-acetyl tryptophanate and then subjected to viral inactivation procedures, including pasteurisation at 60 °C.
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A very similar product also manufactured by GE Healthcare is Omnipaque (Iohexol as the main drug substance). Iodixanol is also the active ingredient in a number of 'cushion' products used during the centrifugation of stallion semen. It is layered underneath the extended stallion semen allowing for a higher g force to be used with less sperm damage and better recovery rates. Post centrifugation the supernatant above and the cushion below is removed, leaving a concentrated sperm pellet in the conical tube.
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This process flow diagram shows how monoclonal antibodies are typically purified at industrial scale. The first reference in the literature to a commercially available protein A chromatography resin appeared in 1976. Today, chromatographic separation using protein A immobilized on porous substrates is the most widely established method for purifying monoclonal antibodies (mAbs) from harvest cell culture supernatant. The choice of protein A as the preferred method is due to the high purity and yield which are easily and reliably achieved.
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The floating layer is generally removed by means of an overflow weir or similar removal method. The aggregated flocculent mass settles either in the reaction vessel or in subsequent settling tanks due to gravitational force. Following removal to a sludge collection tank, it is typically dewatered to a semi-dry cake using a mechanical screw press. The clear, treated (supernatant) water is typically then pumped to a buffer tank for later disposal and/or reuse in the plant's designated process.
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A three hundred milligram soybean powder specimen is mixed with a twenty milliliter compound including 0.5 M NaCl, 0.5% SDS, 20 mM Tris-HCl (pH 7.5), and 2% 2-ME. The compound is then shaken at room temperature for 16 hours for abstraction. The abstract is centrifuged for 30 minutes at twenty thousand gram, then the supernatant is selected by a 0.8-μm microfilter paper. The protein substance from the initial abstract is inspected with a 2-D Quant Kit.
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Another process in common use is to rub a rod on the side of a glass vessel containing the solution to release microscopic glass particles which can act as nucleation centres. In industry, centrifugation is used to separate the crystals from the supernatant liquid. Some compounds and mixtures of compounds can form long-living supersaturated solutions. Carbohydrates are a class of such compounds; The thermodynamic barrier to formation of crystals is rather high because of extensive and irregular hydrogen bonding with the solvent, water.
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Mass spectrometry analysis is integral to secretomics. Serum or supernatant containing secreted proteins is digested with a protease and the proteins are separated by 2D gel electrophoresis or chromatographic methods. Each individual protein is then analyzed by mass spectrometry and the peptide- mass fingerprint generated can be run through a database to identify the protein. Stable isotope labeling by amino acids in cell culture (SILAC) has emerged as an important method in secretomics – it helps to distinguish between secreted proteins and bovine serum contaminants in cell culture.
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This material is often described as sewage fungus but true fungal communities are relatively uncommon. The combination of wastewater and biological mass is commonly known as mixed liquor. In all activated sludge plants, once the wastewater has received sufficient treatment, excess mixed liquor is discharged into settling tanks and the treated supernatant is run off to undergo further treatment before discharge. Part of the settled material, the sludge, is returned to the head of the aeration system to re- seed the new wastewater entering the tank.
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Research has been done investigating the use of diabodies to get around this limitation. Proteins in the supernatant or on the outside of the cell membrane can be bound by the antibodies; this allows for living cells to be stained. Depending on the fixative that is being used, proteins of interest might become cross-linked and this could result in either false positive or false negative signals due to non-specific binding. An alternative approach is using recombinant proteins containing fluorescent protein domains, e.g.
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Therefore, as a cell line (e.g., hybridoma) expands and expresses a target protein, that protein remains within the EC space and is not flushed out. At a given time point (or continually during the culture), the harvest supernatant (product) is collected, clarified and refrigerated for a future downstream application. Smaller hollow fiber bioreactors are often used for selection and optimization of cell linesGramer, MJ. Britton TL. Selection and Isolation of Cells for Optimal Growth in Hollow Fiber Bioreactors Hybridoma 2000. 19(5):407-412.
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Predation by bacterial-feeding nematodes was shown to influence nitrogen availability and plant growth. There was also an increase in the populations of bacteria to which nematodes were added. Predation upon Pseudomonas by amoeba shows predators are able to upregulate toxins produced by prey without direct interaction using supernatant. The ability of predators to control the expression and production of biocontrol agents in prey without direct contact is related to the evolution of prey species to signals of high predator density and nutrient availability.
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The Rapide system allowed without any transfusor apparatus and only with the needle and filter connected carrying bottles, transfusion be held even in the firing line. Blood bottles were stored at 2 °C up to 15 days before use is checked that there is a clear interface between the red cell and plasma package and it maintained its yellowing. Bottles with hemolytic supernatant were discarded. If the appearance of the bottle was right, it was heated in a water bath prior to administration to the patient.
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Supernatant from cells grown in normal medium and cells grown in medium with stable-isotope labeled amino acids is mixed in a 1:1 ratio and analyzed by mass spectrometry. Protein contaminants in the serum will only show one peak because they do not have a labeled equivalent. As an example, the SILAC method has been used successfully to distinguish between proteins secreted by human chondrocytes in culture and serum contaminants. An antibody microarray is a highly sensitive and high-throughput method for protein detection that has recently become part of secretomic analysis.
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A stirring rod is used for mixing liquids, or solids and liquids. Stir rods are used as part of proper laboratory technique when decanting supernatants because the contact helps to negate the adhesion between the side of the glassware and the supernatant that is responsible for the liquid running down the side. Using a stir rod also grants more control over the rate of flow, which is important in cases where chemicals may react violently. This process is also used to pour a large-mouthed flask or beaker into a test tube.
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The motilities of Pseudomonas, Vibrio and Leptospira strains were also severely disrupted by lactose utilization by L. lactis. Using Salmonella flagellar as the experimental group, Nakamura’s team found that a product of lactose fermentation is the cause of motility impairment in Salmonella. It is suggested that the L. lactis supernatant mainly affects Salmonella motility through disturbing flagellar rotation but not through irreversible damage against morphologies and physiologies. Lactose fermentation by L. lactis produces Acetate that reduces the intracellular pH of Salmonella, which in turn slow down the rotation of their flagella.
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A high laminar shear enhances acute endothelial cell response to interleukin-1β in naïve or shear-conditioned endothelial cells as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin down regulation to protect against chronic inflammation. Phytoestrogens, plant compounds with estrogen-like biological activity, such as genistein, formononetin, biochanin A and daidzein, as well as a mixture of these phytoestrogens were found able to reduce E-selectin as well as VCAM-1 and ICAM-1 on cell surface and in culture supernatant.
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It is important to know how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin 18. However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available. These include absence of apoptotic markers (caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation) and differential kinetics of cell death markers (phosphatidylserine exposure and cell membrane permeabilization). A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references.
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Following centrifugation, the supernatant is discarded and the sediment is vortexed. A drop of sediment is placed into an 8 ml Hettich cytocentrifuge chamber prefilled with 2 ml of CytoRich Yellow (TriPath Imaging), a proprietary Saccomanno-like fixative that prevents dehydration and collapse of 3-dimensional structures when slides are air-dried, and then spun onto adhesive-coated slides. Advantages include batch processing and reusability of its funnel assembly, which decreases the bulk of disposable plastic that can significantly impact the environment as well as add to the cost of individual tests.
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Peptidyl-Asp metalloendopeptidase (, endoproteinase Asp-N, peptidyl-Asp metalloproteinase) is an enzyme. This enzyme catalyses the following chemical reaction : Cleavage of Xaa-Asp, Xaa-Glu and Xaa- cysteic acid bonds =History= Peptidyl-Asp metalloendopeptidase was first discovered when it was isolated from the supernatant of Pseudomonas fragi. Originally, it was thought that this bacteria produced only a single proteinase, but later it was discovered that P. fragi can produce more than one proteinase species. This is of interest due to the fact that one of the mutants identified has a cleavage specificity that was previously unknown and can be used in protein sequencing.
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Sludge Dewatering occurs at the site where alum (aluminum sulfate) sludge from sedimentation basin, and spent lime from CT basin and reservoirs is then pumped to sludge drying lagoons. Filter wash water lagoons allow for settling and evaporation. The supernatant of this process is historically discharged to the headworks or sanitary sewer. The EA Fairbairn WTP has three sludge drying lagoons and two filer wash lagoons that are all concrete lines, sludge is removed from these lagoons when they are no longer free draining, and further dried to a solids content of 20-50% before disposal.
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Sublimation of a carbene Stable carbenes are very reactive, and so the minimum amount of handling is desirable using air-free techniques. However, provided rigorously dry, relatively non-acidic and air- free materials are used, stable carbenes are reasonably robust to handling per se. By way of example, a stable carbene prepared from potassium hydride can be filtered through a dry celite pad to remove excess KH (and resulting salts) from the reaction. On a relatively small scale, a suspension containing a stable carbene in solution can be allowed to settle and the supernatant solution pushed through a dried membrane syringe filter.
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FSEC is typically used to compare membrane protein orthologs or screen detergents to solubilize specific membrane proteins in. For fluorescence- detection size-exclusion chromatography-based thermostability assay (FSEC-TS) the samples are heated in the same manner as in FastPP and CETSA and following centrifugation to clear away precipitate the supernatant is treated in the same manner as FSEC. Larger aggregates are seen in the void volume while the peak height for the protein of interest decreases when the unfolding temperature is reached. GFP has a Tm of ~76 °C so the technique is limited to temperature below ~70 °C.
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Osmotic shock or osmotic stress is physiologic dysfunction caused by a sudden change in the solute concentration around a cell, which causes a rapid change in the movement of water across its cell membrane. Under conditions of high concentrations of either salts, substrates or any solute in the supernatant, water is drawn out of the cells through osmosis. This also inhibits the transport of substrates and cofactors into the cell thus “shocking” the cell. Alternatively, at low concentrations of solutes, water enters the cell in large amounts, causing it to swell and either burst or undergo apoptosis.
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The S9 fraction is the product of an organ tissue homogenate used in biological assays. The S9 fraction is most frequently used in assays that measure the metabolism of drugs and other xenobiotics. It is defined by the U.S. National Library of Medicine's "IUPAC Glossary of Terms Used in Toxicology" as the "Supernatant fraction obtained from an organ (usually liver) homogenate by centrifuging at 9000 g for 20 minutes in a suitable medium; this fraction contains cytosol and microsomes." The microsomes component of the S9 fraction contain cytochrome P450 isoforms (phase I metabolism) and other enzyme activities.
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These clumps will then settle to the bottom of the solution tube following centrifugation, carrying the protein of interest. The proteins that are not needed will be found in the supernatant, which can be physically separated from the spherical aggregates. To ensure that there are few impurities in the ELP-protein complex isolated, the solution can be cooled below the Tt, enabling the ELPs to once again assume their linear structure. From this point, hot and cold centrifugation cycles can be repeated, and then the protein of interest can be eluted from the ELPs via the addition of a salt.
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This cleavage generates new 3' termini, which are then dephosphorylated, resulting in 3'OH ends that can be used as starting points for a new step of extension. This results in the elongation of the damaged strand, from the damaged region towards the bounded bead: while the new DNA molecule is synthesised, the original fragment is displaced. As a result, the dsDNA molecules newly formed no longer contain the adaptor bound to the beads, leaving in the supernatant a dsDNA library of the strands that originally harboured deaminated cytosines, available for further amplification and sequencing. The undamaged DNA template fraction remains attached to the paramagnetic beads.
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A typical complete trickling filter system Image 1. A schematic cross-section of the contact face of the bed of media in a trickling filter Broken trickling filter unit at the sewage treatment plant in Norton, Zimbabwe, showing importance of maintenance to prevent structural failure Typically, settled sewage flow enters at a high level and flows through the primary settlement tank. The supernatant from the tank flows into a dosing device, often a tipping bucket which delivers flow to the arms of the filter. The flush of water flows through the arms and exits through a series of holes pointing at an angle downwards.
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Higher hydrostatic pressure at the axial end (media entering the fiber lumen) compared to the distal end of the bioreactor creates a Starling flow in the EC space, which is similar to what is observed in the body. This phenomenon also creates a nutrient-rich axial region and a nutrient-depleted distal region within the bioreactor. By incorporating EC cycling, the effects of Starling flow are eliminated and the entire bioreactor becomes nutrient-rich and optimized for cell growth. Optimal IC and EC space perfusion rates must be achieved in order to efficiently deliver media nutrients and growth supplements, respectively, and to collect supernatant.
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If the protein of interest is not secreted by the organism into the surrounding solution, the first step of each purification process is the disruption of the cells containing the protein. Depending on how fragile the protein is and how stable the cells are, one could, for instance, use one of the following methods: i) repeated freezing and thawing, ii) sonication, iii) homogenization by high pressure (French press), iv) homogenization by grinding (bead mill), and v) permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g. lysozyme). Finally, the cell debris can be removed by centrifugation so that the proteins and other soluble compounds remain in the supernatant.
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This is because sepharose beads must be concentrated at the bottom of the tube by centrifugation and the supernatant removed after each incubation, wash, etc. This imposes absolute physical limitations on the process, as pellets of agarose beads less than 25 to 50μl are difficult if not impossible to visually identify at the bottom of the tube. With magnetic beads, there is no minimum quantity of beads required due to magnetic handling, and therefore, depending on the target antigen and IP antibody, it is possible to use considerably less magnetic beads. Conversely, spin columns may be employed instead of normal microfuge tubes to significantly reduce the amount of agarose beads required per reaction.
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NUN buffer is a solution that makes it possible to purify proteins located in the nucleus of eukaryotic cells. Although other procedures are available they result in loss of albumin D-box binding protein (DBP) which is unwanted if nuclear signal pathways are to be investigated. Therefore, a new extraction procedure was developed in 1993 to increase recovery of nonhistone proteins using a (NUN) solution containing 0.3 M NaCl, 1 M urea, and 1% nonionic detergent Nonidet P-40, which destabilize salt bridges, hydrogen bonds, and hydrophobic interactions, respectively; resulting in a disruption of interaction between proteins and DNA. By incubating nuclei in NUN buffer and centrifuging the solution, the supernatant will therefore contain nuclear proteins.
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Secondly, Sutherland initially believed that there was something vital about the intact cell, and that disrupting its structure would not produce any hormonal effect. However, after some debate, Rall had convinced Sutherland to use liver homogenates. Once they had witnessed nearly a doubling of the rate of LP activation, they knew this belief in that keeping cells intact was crucial to studying the effects of hormones was not necessarily true, at least in this case. Finally, Sutherland had decided to ignore Jacques Berthet's request to conduct the same experiment using proper lab technique, specifically the Lehninger Hard Pour, where the supernatant material was decanted by pouring the liquid into another test tube once the particulate fraction reached the top of the original tube.
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Cell cultures from the patient's bone morrow grew an abnormally high percentage (52%) of eosinophil Colony-forming units (CFUs). Nine of 25 cell clones derived from the patient's blood T cells stimulated abnormally high (>60%) eosinophil CFUs when incubated with bone marrow cells taken from a non-identical donor; supernatant fluid taken from the patient's T cells was also active in inducing eosinophil CFUs from the non-identical donor's bone marrow cells. Immunophenotyping of these eosinophil CFU-stimulating T cells indicated that they expressed the CD4 but not CD8 cell surface Cluster of differentiation antigen, suggesting that they were cytokine-secreting helper T cells. Characterization of the T cell receptor on these T cell's revealed several patterns of rearrangement in the receptor's β chains.
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These beads are coated to bind to targeted biomolecules, gently separated and goes through multiple cycles of washing to obtain targeted molecules bound to these super paramagnetic beads, which can differentiate based on strength of magnetic field and targeted molecules, are then eluted to collect supernatant and then are able to determine the concentration of specifically targeted biomolecules. The technique of immunomagnetic separation (IMS) obtains certain concentrations of specific molecules within targeted bacteria. A mixture of cell population will be put into a magnetic field where cells then are attached to super paramagnetic beads, specific example are Dynabeads (4.5-μm), will remain once excess substrate is removed binding to targeted antigen. Dynabeads consists of iron- containing cores, which is covered by a thin layer of a polymer shell allowing the absorption of biomolecules.
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These lentiviral vectors are able to efficiently transduce a broad range of cell types and stably integrate into the genome of dividing and non-dividing cells. Third generation lentiviral particles are produced by co-transfecting 293T human embryonic kidney (HEK) cells with: # two packaging plasmids, one encoding Rev and the other Gag and Pol; # an interchangeable envelope plasmid that encodes for an envelope glycoprotein of another virus (most commonly the G protein of vesicular stomatitis virus (VSV-G)); # one or two (depending on the applied library) transfer plasmids, encoding for Cas9 and sgRNA, as well as selection markers. The lentiviral particle-containing supernatant is harvested, concentrated and subsequently used to infect the target cells. The exact protocol for lentiviral production will vary depending on the research aim and applied library.
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In a liquid medium with few or no expected organisms, from an area that is normally sterile (such as CSF, blood inside the circulatory system) centrifugation, decanting the supernatant and using only the sediment will increase the chance to grow and isolate bacteria or the usually cell-associated viruses. If one expects or looks for a particularly fastidious organism, the microbiological culture and isolation techniques will have to be geared towards that microbe. For example, a bacterium that dies when exposed to air, can only be isolated if the sample is carried and processed under airless or anaerobic conditions. A bacterium that dies when exposed to room temperature (thermophilic) requires a pre-warmed transport container, and a microbe that dries and dies when carried on a cotton swab will need a viral transport medium before it can be cultured successfully.
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Subsequently, the sterility of the blood contained in the flask was checked by withdrawing a sample was seeded on agar tubes and the supernatant is used to recheck the blood group of the flask. However, errors in blood grouping because, among other things, the quality of the reagents were prepared in the same service, it is advised to perform before transfusion biological test Ochlecker: inject 5-10 ml blood, wait 10–15 minutes and continue transfusion if there had been no symptoms. If extraction is believed to be correct, the blood of six donors from the same group was mixed in an Erlenmeyer flask of two litres, by passing it through a filter consisting of silk fabric with a pore size of about 250 microns. This is managed to remove clots and aggregates which might have formed during bleeding.
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Compared with previous method, a heating process is involved in current abstraction technique to investigate soybean protein existing in brewed products. Since the heating process can deactivate the microbial proteolytic enzymes, the current abstraction technique can be used to disclose soybean protein in brewed soybean products. The heating abstraction technique can be demonstrated as the following. To produce the good dispersibility for the specimen in the extraction buffer to carry out the heating process, 19mL of abstraction buffer is mixed with five glass beads in five millimeter diameter and 1 g of food homogenate. At 5, 15 and 60 min variable time, the mixture is abstracted under 25, 40, 60, 80 and 100 ° variable temperature through the heating in a water bath followed by every 5 minutes vortexing. Food abstractions generated through the previous and the current technique are centrifuged for 20 minutes at three thousand gram, then the supernatant is filtered off by a filter paper.
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