Methods Cell permeabilization

Target access Try several methods to permeabilize cells, each method having a different basis. 

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... 

Methods for Permeabilizing Cells 116 Examples of Intracellular Staining 118... 

Secondary metabolites produced by plant cell culture are commonly accumulated in the cells. With few exceptions such as Capsicum frutescens, Thalictrum minus (9) and Vanilla planifolia (Knorr, D. and Romagnoli, L., Univ. of Delaware, unpublished data) cultures, which release valuable compounds such as capsaicin, berberine and vanillin, respectively, into the medium, procedures to induce product release are required to develop continuous production processes. Reported permeabilization methods include treatment with dimethylsulfoxide (DMSO), isopropanol, toluene, phenethyl alcohol or chloroform (9, 28). But as Fontanel and Tabata (9) pointed out, such treatments with organic solvents are severe and other methods of permeabilization need to be developed. 

Because intact cells are impermeable to macromolecules, they have to be permeabilized before the NPC can be labeled with antinucleoporin antibodies. Alternatively, isolated nuclei or nuclear envelopes can be used. Therefore we first describe the various methods for cell permeabilization and nuclear isolation and then summarize the procedures used for immunolabeling. 

For immunolabeling purposes as well as nucleocytoplasmic transport studies, monolayers of cultured cells can be easily permeabilized by treatment with digitonin (Adam et ai, 1990). The rationale behind the method is that digitonin more or less selectively attacks membranes containing cholesterol (such as the plasma membrane) but not membranes containing no or very little cholesterol (such as the nuclear membranes). We use the following method to permeabilize 3T3 cells ... 

The following method of cell permeabilization is a modification of the method originally described by Adam et al. (1990). We have successfully applied it to a number of different cell types to give permeabilized cells competent for nuclear protein and snRNP import in vitro. It has the advantage that large numbers of permeabilized cells can be produced in a single batch and stored frozen in small aliquots for transport experiments. This eliminates the variation that can arise when cells are permeabilized on cover slips and each cover slip is then used for a single data point in an assay. 

Adam, S. A., Sterne-Marr, R., and Gerace, L. (1991). In vitro nuclear protein import using digitonin permeabilized mammalian cells. Methods Cell Biol. 35, 469-481. 

Diaz, R., and Stahl, P. D. (1989). Digitonin permeabilization procedures for the study of endosome acidification and function. Methods Cell Biol. 31, 25-43. 

Chaotropes are strong protein denaturants and induce cell permeabilization by weakening the hydrophobic interactions of the membrane. In case of bacterial cells, chaotropic agents also inhibit the assembly of cross-linked peptidogly-cans in the cell wall. Another common method of disrupting bacterial cell walls is enzymatic digestion using lysozyme, which hydrolyzes the polysaccharide component of the cell wall. 

Exposure to active oxygen results in a transient increase in poly(ADP-ribose) concentrations. We measured the AO induced increase in poly(ADP-ribose) in monolayer cultures of JB6 clone 41 by the fluorimetric method of Jacobson et al. (31) which avoids cell permeabilization. At a concentration of 50 pg X/ml plus 5 pg XO/ml, which generates within 30 min a concentration of 5 x 10 M Oj, the level of poly(ADP-ribose) reached 70 pmoles eRAdo per 10 cells. After this maximum, poly(ADP-ribose) concentrations slowly decreased and had not returned to control levels after 2 hrs. Because of tiieir inhibitory action on AO induced DNA breakage we studied the effects of the Ca -chelator Quin 2 and the Fe-chelator o-phenanthroline on poly(ADP-ribose). Both drugs completely prevented the increase in poly(ADP-ribose) by AO at relatively low doses. In contrast, experiments carried out in parallel indicated that... 

For cell permeabilization various methods can be applied. Treatment with L-a-lysophosphatidylcholine (LPC) has proven to be a cheap, easy, and versatile way to perforate cells. If soluble cytoplasmic proteins need to be labeled other reagents generating smaller holes, such as Staphylococcus aureus a-toxin, can be used. If only the plasma membrane needs to be permeabilized the LPC concentration should be adjusted to obtain 95% trypan blue-stainable cells. 

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immuno-localization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immuno-localization at the level of light microscopy. 

Schmid, 1., Uittengobaart, C. H., and Giogi, J. V. (1991) A gentle fixation and permeabilization method for combined cell-snrface and intracellular staining with improved precision in DNA qnantification. Cytometry 12, 279-285. 

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... 

Suehiro, J., Shutou, M., Hatano, T., and Hara, M. (2003). High sensitive detection of biological cells using dielectrophoretic impedance measurement method combined with electro-permeabilization. Sens. Actuators B Chem. 96,144-151. 

Despite the harshness of these reagents (see Note 1), the organic solvents are the simplest and most rapid method to fix and permeabilize cells. Methanol is preferable to ethanol in many cases. The basic fixation procedure outlined below is suitable for the detection of total PCNA within the cell and for Ki-67 in most cells. 

It is possible to permeabilize the outer membrane of normal cells (with detergent or alcohol) in order to allow propidium iodide to enter the nuclei. If we then treat the normal cells with RNase in order to ensure that any fluorescence results from their DNA content (without a contribution from double-stranded RNA), we find that the nuclei fluoresce red with an intensity that is more or less proportional to their DNA content. By the use of a red filter and a linear amplifier on the photomultiplier tube, we can detect that red fluorescence. The channel number of the fluorescence intensity will be proportional to the DNA content of the cells. The method is simple and takes about 10 minutes. Flow cytometric analysis of the red fluorescence from the particles in this preparation of nuclei from normal, nondividing cells will result in a histogram with a single, narrow peak (see the first histogram in Fig. 8.1) all the particles emit very nearly the same amount of red fluorescence. This supports our knowledge that all... 

Dual staining of cells can also be used to look at DNA and protein markers simultaneously. Using methods similar to those described in the previous chapter for looking at cytoplasmic proteins simultaneously with surface membrane proteins, cells can be stained for surface proteins, then fixed and permeabilized, and then stained for DNA. Figure 8.16 shows the way that this type of technique can be used to permit the cell cycle analysis of subpopulations of cells independently. In this particular example, it can be seen that, after treatment with the mitogen phytohemagglutinin, it is the CD8-positive cells more than the CD8-negative cells that have been induced to enter S phase. 

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