Permeabilized cells

Figure 3. The binding and dissociation of FU EP and receptor on permeabilized neutrophils at 37 C The data are plotted as the specific binding (pmoles FLPEP bound/lO cells) on a log plot versus time. 10 permeabilized cells/mL were exposed to 1 nM FLPEP at time 0. After IS or 120 s, antibody to fluorescein was added to each sample. 60 seconds later, 10- M GTP yS was added. (Reproduced with permission from reference 22. Copyright 1987 Journal of Biological Chemistry.)...

Figure 4 shows an analysis of binding and dissociation of FLPEP in permeabilized cells in the absence (top panel) and the presence of GTPyS (lower panel). Dissociation is initiated, as indicated, by the addition of a receptor antagonist, tBoc-phe-leu-phe-leu-phe. The solid line is a fit to the data. The data from this experiment have... 

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. 

Eberhard, D. A., Cooper, C. L., Low, M. G. and Holz R. W. Evidence that the inositol phospholipids are necessary for exocytosis loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATR Biochem. J. 268 15-25, 1990. 

Taylor The experimental observation from which this comes is as follows. First, we pretreat the permeabilized cells in our superfusion apparatus with 100 /rM Ca2+ for about a second, to drive the cells to the state where they can no longer respond to InsP3 (even at heroic concentrations). Next, we wash out the Ca2+ to a low cytosolic level of 200 nM. Then we look for how long it takes for the normal response to a maximal concentration of InsP3 to recover. That takes a long time. 

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

Alcohol, protease, or lipase treatment may be used to permeabilize cells. Proteolytic digestion is best for negation of crosslinking. 

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

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]. 

An alternative explanation that may also account for the inability of the C-terminal peptide to compete for cell-surface interactions is that its binding site is located not on the extracellular domain of the receptor, but rather on the intracellular domain. The primary differences between the cell-surface form of the IFN-y receptor and (2) the accessibility of the recombinant receptor s cytoplasmic domain. A synthetic peptide corresponding to the membrane proximal region of the cytoplasmic domain of the murine IFN-y receptor was able to bind IFN-y and specifically compete with the binding of IFN-y (95-133) to fixed/permeabilized cells [33]. 

Preembedding hybridization where permeabilized cells or vibratome sections are hybridized, and then postfixed and embedded as described in Section 3.1.2. 

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. 

In a simple procedure for DNA cloning, an autonomously replicating plasmid and insert DNA are cut with a restriction enzyme and then the pieces are annealed and covalently joined by the action of DNA ligase. The resulting recombinant molecules are then transfected into E. coli, where they replicate. When plasmid vectors are used, a population of permeabilized cells is bathed in the plasmid DNA containing the inserted DNA. Because only a small number of cells become transfected by this procedure, a way to se-... 

SDS disrupts noncovalent interactions between subunits of a protein, so if a protein has two subunits, two bands will appear. In the absence of SDS, only one band will appear. This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. This property of SDS may be responsible for protein denaturation. It should be noted that SDS also permeabilizes cells for antibody access to intracellular epitopes. 

Grube, K., Kupper, J.H. and Burkle, A. (1991) Direct stimulation of poly(ADP-ribose) polymerase in permeabilized cells by double-stranded DNA oligomers. Anal. Biochem., 193, 236-239. 

Wilson, G.L., Dean, B.S., Wang, G. and Dean, D.A. (1999) Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J. Biol Chem., 274, 22025-22032. 

Figure 12.5 Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and permeabilized with digitonin as described in Wilson et al., 1999. Fluorescein-PNA-labeled plasmids (containing the SV40 enhancer, 4.2 kb) or rhodamine-labeled BSA-NLS peptide conjugates were incubated with the cells for four hours at which time they were viewed by fluorescence microscopy. With no additions, neither DNA nor protein was imported, but in the presence of nuclear and cytoplasmic extracts both substrates localized to the nuclei. While plasmids containing the SV40 enhancer were taken up by the nuclei, those lacking the sequence were excluded. The remaining panels demonstrate the need for both the import machinery (importins and Ran) and a source of adapter proteins (nuclear extract) for plasmid nuclear entry, but not for protein nuclear localization.

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