Permeabilization applications

Adtveoitages. The antibody technology is remarkably convenient. It requires only that a significant fraction of FLPEP be receptor bound ( 10%). The assay can be set up in a few minutes and is applicable to membranes, permeabilized cells, cells, or any other preparation in which the receptor concentration is in the range of 1 niV or greater. We use this assay routinely to analyze ligand dissociation kinetics. 

In the interdisciplinary field of biophysics and biotechnology, the bioeffects of electric field have received considerable interest for both fundamental studies on these interaction mechanisms and potential application. However, the effects of pulsed electric field (PEF) on secondary metabolism in plant cell cultures and fermentation processes have been unknown. Therefore, it would be very interesting to find out whether PEF could be used as a new tool for stimulating secondary metabolism in plant cell cultures for potential application to the value-added plant-specific secondary metabolite production. Furthermore, if the PEF permeabilization and elicitation are discovered in a cell culture system, the combination of... 

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

PANCREAS Membrane potential measurements in pancreatic /3 cells with intracellular microelectrodes, 192, 235 stimulation of secretion by secretagogues, 192, 247 pancreatic secretion in vivo, perfused gland, and isolated duct studies, 192, 256 dispersed pancreatic acinar cells and pancreatic acini, 192, 271 permeabilizing cells some methods and applications for the study... 

Furthermore, the type of enzyme formulation (free enzyme, immobilized enzyme, or whole cells) plays a key role in determining the progress of the overall reaction. For most applications, lyophilized enzyme powders have been used with good results presumably they dissolve into the liquid phase. When poorly soluble products are formed, the enzyme can be recovered by washing with water [52]. For co-factor-dependent reactions permeabilized cells may be used [44]. When using immobilized enzymes, it has been demonstrated that the chemical nature and the pore size of the support are very important parameters to consider [8, 41]. 

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

Application of an electric field to lipid bilayers such as those found in cellular membranes causes short-term depolarization of the membrane and formation of pores and other structural changes [17]. These so-called electropores allow the uptake of hydrophilic macromolecules such as plasmid DNA, siRNA, or proteins that are otherwise unable to diffuse passively through this highly regulated barrier. The use of high-voltage electrical pulses to permeabilize cell membranes was first described as a tool to deliver DNA into mammalian cells in 1982 (Wong and Neumann 1982 Neumann et al. 1982). In cuvette-based methods, cells are... 

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Integrated bioprocesses can be used to enhance the production of valuable metabolites from plant cell cultures. The in situ removal of product during cell cultivation facilitates the rapid recovery of volatile and unstable phytochemicals, avoids problems of cell toxicity and end-product inhibition, and enhances product secretion. In situ extraction, in situ adsorption, the utilization of cyclodextrin, and the application of aqueous two-phase systems have been proposed for the integration of cell growth and product recovery in a bioreactor. The simultaneous combination of elicitation, immobilization, permeabilization, and in situ recovery can promote this method of plant cell culture as a feasible method to produce various natural products including proteins. 

This protocol requires fixation and permeabilization of the cells, therefore it is only applicable to ligands for which this is possible. GFP-tagged dominantnegative dynamin constructs are, however, available (19) and should enable the use of live cells. 

The fixation conditions used to prepare cells for antibody application are assumed to preserve the distributions of the protein(s) being examined (2). Soluble proteins can be redistributed into inappropriate locations and can be differentially extracted from native locations during the permeabilization and fixation of the cells before antibody application (3,4)- Further, no cell aggregation or alteration of the intracellular antigenicity should occur in the permeabilization/fixation treatment. The fixation/stain methodology, with and without permeabilization, can be accomplished in various ways depending on the exact site of the organelle or cell constituent to be stained. 

When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 ng/ml. Most cells will become perme-abilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP. One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi ef al., 1989)). ATP depletion will always be found in cells that have been permeabilized. 

Lind I, Ahnert-Hilger G, Fuchs G et at. (1987) Purification of alpha-toxin from staphylococcus aureus and application to cell permeabilization. Anolyt. Biochem. 164 84-89. 

Another study tested to what extent antibiotics affect the membrane integrity of Escherichia coli and hence inhibit the ChemChrome V6 labelhng. Inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at concentrations below the MIC but not for antibiotics with other mechanisms of action e. g. 6-lactams. As an application, colistin could be determined in milk by measuring the decrease in the number of labelled Escherichia coli cells relative to the initial number that had been added to the milk (D Haese and Nelis 2000). 

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