Cell permeabilization

Procedures of the beta-galactosidase activity measuring using colour reaction with ONPG and X-Gal without cells permeabilization were developed and the detection limit at the level of 4 ppb has been achieved. The influence of the foreign ions (phosphate, sulphate, carbonate et. al) was studied. 

Cell permeabilization and uptake of anti-sense peptide-peptide nucleic acid (PNA) into Escherichia coli. J. Biol. Chem. 2002 277 7144-7147. 

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. 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

Brearley, C.A. Hanke, D.E. Metabolic relations of inositol 3,4,5,6-tetrakis-phosphate revealed by cell permeabilization. Identification of inositol 3,4,5, 6-tetrakisphosphate 1-kinase and inositol 3,4,5,6-tetrakisphosphate phosphatase activities in mesophyll cells. Plant Physiol., 122, 1209-1216 (2000)... 

It should also be noted that denaturing agents make cells permeable, facilitating antibody penetration. Moreover, these agents are used usually in combination with microwave heating. Therefore, the role of these agents in epitope retrieval needs to be explained in the context of their role in cell permeabilization as well as of the influence of elevated temperatures. 

PNA-peptide conjugates should preferably be used at low concentrations (below 2.5 fj /) to avoid cell permeabilization. 

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. 

Take the microwell plate to a light microscope. Carefully remove the trypan blue working solution till a thin film is left on the cells. Permeabilized cells will appear blue. 

Clostridial toxins are commercially available from Calbiochem and from Sigma. Antibodies against VAMPs, SNAP-25 and syntaxins can be obtained from Chemicon and from Sigma. No special material is required in addition to that given for cell permeabilization, except for a plastic container that should resist a temperature of 120°C. This con-... 

Prepare all buffers required for cell permeabilization and for secretion measurements (see Section 16.2.3). 

In addition to the buffers needed for cell permeabilization prepare Solution LC supplemented with the required amount of TeTx. If the whole toxin (heavy plus light chains) is used, the catalytic subunit must be separated from the heavy chain by dithiothreitol (DTT) treatment. We usually incubate a 200-fold concentrated toxin in LC supplemented with DTT (final concentration 10 mM) for 30 min at 37°C. If the light chain is used this activation step is unnecessary. 

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. 

Permeabilized cells allow the study of intracellular processes in situ under conditions which are believed to be close to the physiological situation in intact cells. Permeabilization by bacterial pore-forming toxins, alpha-toxin and streptolysin O (SLO) is now a widely accepted approach in the functional analysis of intracellular organelles. 

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. 

Triton skinned and glycerinated fibers have been very valuable in demonstrating that phosphorylation and dephosphorylation of MLC is sufficient to induce contraction and relaxation (see Section III.B). These preparations have also been used to study the influence of ionic strength (Arheden et al., 1988 Gag-elmann and Guth, 1985), free Mg + (Arner, 1983 Bar-sotti et al., 1987), pH (Mrwa et al., 1974), inorganic phosphate (Schneider et al., 1981), nucleotides such as ATP and ADP (Arner and Hellstrand, 1985) on isometric force development, shortening velocity, and ATP turnover. Some of these experiments have also been carried out in smooth muscle fiber bundles and single smooth muscle cells permeabilized with saponin, (3-escin, or a-toxin (Saida and Nonomura, 1978 lino, 1981 Warshaw et al., 1987 Crichton et al., 1993). 

Most immunofluorescence protocols involve fixation of cells at a specific time point of activation to preserve the distribution of intracellular proteins, followed by cell permeabilization to allow the entry of the antibody into the cell. The primary antibody can either be directly tagged with a fluorophor or, in the indirect approach, a secondary anti-lgC or anti-lgM antibody is tagged. The direct approach is simpler and less prone to background problems. In contrast, the indirect approach offers the advantage of widely available labeled antibodies and the increased sensitivity that results from amplification of the signal (for more details, see Harlow and Lane, 1988). 

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