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Downstream Processing
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
Density gradient centrifugation is similar to differential sedimentation in that the principle is to exploit centrifugal force to separate feed components on the basis of particle size or density. However, the distinguishing feature is that the medium in the centrifuge tube is heterogeneous, and the density of the solution increases along the axis of rotation. This is usually achieved by varying the concentration of solute along the centrifuge tube prior to sample loading. As well as pelleting very dense particles, density gradient centrifugation also achieves the separation of particles within the density range of the solution into different bands. In rate zonal centrifugation, all particles will eventually form a pellet so the run must be interrupted to isolate particles in the desired density range. In isopycnic centrifugation, a very dense solution is used and particles or molecules in the density range will never form a pellet but will remain suspended at a specific distance along the density gradient. Such processes are common in laboratory-scale preparations but are rarely used on an industrial scale.
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Published in Luis Liz-Marzán, Colloidal Synthesis of Plasmonic Nanometals, 2020
Jose M. Romo-Herrera, Ramón A. Alvarez-Puebla, Luis M. Liz-Marzán
The first separation methods employed were chromatography82-84 and size-selective precipitation,85-87 for very small NPs (1–10 nm size range). These methods show great capabilities to improve the size monodispersity but are commonly used in a size regime smaller than the one considered here. Plasmonic building blocks correspond to a larger size regime, for which the reported separation methods can be sorted into three main classes, as a function of their driving force: (i) sedimentation, (ii) electrophoresis, and (iii) density gradient centrifugation.
Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining
Published in Environmental Technology, 2022
Kamila Jessie Sammarro Silva, Lyda Patricia Sabogal-Paz
The viability assays were performed over suspended Giardia muris cysts and Cryptosporidium parvum oocysts acquired live. Both suspensions (Waterborne™, Inc, USA) were extracted from experimentally infected Swiss Webster (CFW®) mice and calves, respectively, and purified from feces by sucrose and percoll density gradient centrifugation. The organisms were stored at 4°C in deionized water with penicillin, streptomycin, gentamicin, amphotericin B and 0.01% Tween® 20, following the supplier’s recommendations. Viability tests and slide counts were carried out promptly, in order to prevent die-off of the organisms during the experiments. The (oo)cysts were not subjected to heat killing or any inactivation control in this study. There is often, however, a fraction of non-viable (oo)cysts in purified suspensions, even within early evaluation.
A method for separation and purification of mouse splenocytes by density gradient centrifugation
Published in Preparative Biochemistry & Biotechnology, 2021
Yao Lu, Chenghao Fu, Chao Xia, Shiliang Ma
Efficient techniques are very essential to isolate specific cells for understanding its behaviors. Currently, the methods of cell separation commonly include density gradient centrifugation[8], immunomagnetic bead enrichment[9] and flow cytometry sorting[10]. Immunomagnetic beads and flow cytometry are performed by combining specific-labeled antibody with protein on cell membrane surface to sort cells of interest. Although cells can be enriched with high purity by these two methods, there are also some shortcomings. For example, flow cytometry can cause cells damage deeply and immunomagnetic beads are expensive. In addition, what we often overlook of these methods is that it may alter the cells behaviors or trigger signaling by crosslinking cell surface receptors or lead to cell toxicity by magnetic beads[11–14] due to antigen-antibody combination. Therefore, changes of cell function may be detrimental to anti-tumor immune response and mislead judgments of the facts further. For density gradient centrifugation, Ficoll[15] and Percoll[16] are most widely used to separate cells according to different density of components[17]. Additionally, the separation solutions keep the cells in a mild environment during separation and protect the cell surface receptors from activating. Therefore, this process has minimal impact on the cells and is also beneficial to subsequent research.
Characterization of aerobic granular sludge of different sizes for nitrogen and phosphorus removal
Published in Environmental Technology, 2019
Zhi-Hua Li, Yuan-Mo Zhu, Ya-Li Zhang, Yu-Rong Zhang, Chun-Bo He, Cheng-Jian Yang
DO and pH in the AO-SBR reactor were measured by portable meters (HQ40d, HACH, America and PB-10, Sartorius, Germany). The temperature in the reactor was measured by an alcohol thermometer. The mixed liquid suspended solids (MLSS) of the sludge were measured according to the standard methods (APHA 1998). The morphology of the granules was observed by using either a digital camera or a microscope (Eclipse 90i microscope, Nikon, Japan). The density of aerobic granules was determined by using a sucrose density gradient centrifugation. The porosity of the granules was calculated as follows: (a) Fresh granules were washed three times using PBS. Then they were embedded in an optimal cutting temperature (O. C. T) compound (Tissue-Tek, Sakura, USA) and were frozen at −20°C. (b) Microtome (HM500, Leica, Germany) was used for the cryosection to obtain a 30-µm thick section. (c) The optical porosity was calculated from the microtome sliced photos (NIS-Elements AR 3.0, Nikon, Japan), and at least 15 samples were analysed.