Permeabilization with

FIG. 2. Confocal image of an isolated smooth muscle cell from guinea-pig ileum which has been permeabilized with staphylococcal os-toxin and incubated with 150 /iM Fluo-3 acid which stains the SR. The SR is predominantly localized to the periphery of this type of smooth muscle as seen on the left hand side where the image plane is through the centre of the cell, whereas an extensive network is seen where the image plane is adjacent to the plasma membrane as seen in the right hand portion of the cell. 

In Section 5.03.6.2, a stereoselective synthesis of L-homophenylalanine from the racemic AAacetylated amino acid is described. The authors, however, found that substrate solubility limited the utility of this procedure. Having found an L-N-carbamoylase in Bacillus kaustophilus, they introduced the gene for this enzyme together with that for the N-acyl amino acid racemase from D. radiodurans into E. coli for coexpression. These cells, permeabilized with 0.5% toluene, were able to deliver L-homophenylalanine in 99% yield and were able to be used for multiple reaction cycles. 

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.

Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and 217 permeabilized with digitonin as described by Wilson et al., 1999... 

Fig. 7.2. Dot plots showing the staining of lymphocytes for intracellular interferon-y in conjunction with an outer membrane stain (against CD8) to phenotype the cytokine-producing cells. Cells were stained for CD8 and then fixed with formaldehyde and permeabilized with saponin. The stimulus was PMA-ionomycin. Data courtesy of Paul Wallace.

Cells were then fixed with 75 pL 4% paraformaldehyde-PBS at 37°C for 20 to 30 min, washed (three times with 300 pL) and permeabilized with PBS-0.1 % TritonX-100 (PBST). 

Several organic solvents were investigated with regard to stability and activity of HLADH as well as their influence on the hydrogenase-driven reaction. Hydrophobic solvents such as heptane or toluene proved to be the most suitable solvents for the coupled enzyme-system. Furthermore, it became apparent that nonimmobilized cells, permeabilized with cetyl-trimethylammonium bromide, showed the best results for NADH regeneration. After optimization the conversion in heptane with 10% water yields 99% cyclohexanol by reduction of cyclohexanone. 

Adherent cell line expressing high cytoplasmic levels of the specific antigen that can be accessed following fixation and permeabilization with methanol. Wash the cells with ice-cold DMEM, then add to each well 200 XL of methanol that has been precooled to -70°C by standing in cardice-cthanol. Leave at ambient temperature for 5 min, flick off the methanol, and wash twice with medium containing either 5% PCS (live cells) or PBS-Marvel (fixed cells) and... 

Permeabilization with carboxyfluorescein-diacetate. 6-carboxy-fluorescein-diacetate (CFDA) is a nonfluorescent, nonpolar reagent which enters the cell freely, where it becomes entrapped following enzymatic conversion to the hydrophilic fluorophore 6-carboxyfluorescein (CF, mw 370). Thus, labeled cells can then be replated onto unlabeled test cells to measure dye transfer via gap junction. This method has been successfully used to measure gap junction communication in early embryo cells (Goodall and Johnson, 1982 Kidder et al., 1987), and also to label tumor cells in culture (Price et al., 1995). We tried to extrapolate it for detection of gap junction formation among cultured cells. 

Cells are permeabilized with 0.2 % Triton X-IOO/PBS at room temperature for 5 min. 

Fig. 2. Indirect immunofluorescence labeling of cell-bound component II. Component II (0.5ng/well) was incubated with cultured Vero cells at 37 °C for 7.5 min (a) not permeabilized with Triton X-IOO-PBS and (b) permeabilized with Triton X-100-PBS. For details see text (Section 9.5.4)... 

Ahnert-Hilger G, Bader MF, BhakDai S et al. (1989b) Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocy-toma cells (PC12) by permeabilization with streptolysin O inhibitory effect of tetanus toxin on catecholamine secretion. J. Neurochem. 52 1751 -8... 

Cells are seeded in a microwell plate 2 to 3 days before the experiment to reach about 80 % confluency on the day of the experiment to favour adherence during subsequent manipulations. Remember that certain cell lines have to be cultured on a special matrix. On the day of the experiment cells in a microwell plate are transferred to a 37°C waterbath. The temperature should be checked with a thermometer since variations may influence exocytosis. Cells are first washed with KRH, than permeabilized with SLO in 0.1 iM free Ca ". The cells are exposed to SLO for 7 min. Subsequently, cells are preincubated (if necessary) at 0.1 xM Ca and then shifted to 0.1 M or 10 jxM Ca ". Finally, the supernatant is transferred to microcentrifuge tubes, centrifuged at 10 000 g for 2 min to sediment any contaminating cells thereafter aliquots are used for determination of the secretory product. 

Ahnert-Hilger G, Bhakdi S, Gratzl M (1985) Minimal requirements for exocytosis a study using PC12 cell permeabilized with staphylococcal a-toxin. In J Biol Chem 260 12730-12734. 

Transmitter uptake into secretory vesicles is an ATP-dependent process. So far these studies have been restricted to isolated secretory vesicles, where intracellular substances necessary for regulation may be lost during purification. Neuroendocrine cells or synaptosomes permeabilized with both pore-forming toxins can be used to study the regulation of transmitter storage without the necessity of purifying secretory vesicles. 

PC 12 cells were permeabilized with purified SLO as given in section 18.3.1 of this chapter. Protein content was measured using the BCA-method. Values are the mean of three samples SD. Similar results were obtained using either wild-type SLO fusion protein or its alanine mutant. The assay can also be performed using alpha-toxin for permeabilization. 

Fig. 1. Cultured RBL-2H3 cells unpermeabilized (a) and permeabilized with acetone (b) for 5 min at -20°C. Without permeabilization, there is no immunostaining. Flowever, after permeabilization, the antibody has access to the antigen and the cells immunolabel.

Blocking and permeabilization solution contains 10 mg/ml BSA, 5.0 % normal goat semm to block nonspecific antibody binding and 0.02% sodium azide is used to prevent bacterial growth during incubations all in PBS. Perform permeabilization with 0.2% Triton. Incubate for 1 h. 

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