Permeabilization of cells

Permeabilization of cells with 0.1-0.2% detergent after paraformaldehyde fixation can leave an uneven cytoplasmic distribution of the labeled proteins, and some of the larger proteins are redistributed to the nuclei. Extraction with 1 % de tergent prior to fixation removes most, but not always all of the exogenous proteins from the cell remnants. 

Detergent Detergents, such as saponin, Triton X-100, or NP40, are used to aid in the permeabilization of cell membranes in order to facilitate staining of intracellular proteins. 

Sundaram, J., Mellein, B.R. and Mitragotri, S. (2003) An experimental and theoretical analysis of ultrasound-induced permeabilization of cell membranes. Biophys. J. 84, 3087-3101. 

Plant cell cultures represent a potentially rich source of secondary metabolites of commercial importance and have been shown to produce them in higher concentrations than the related intact plants. However, plant cell cultures often produce metabolites in lower concentrations than desired and commonly store them intracellularly. These limitations can be overcome by product yield enhancement procedures, including immobilization of cultured cells, and permeabilization, or ideally using a combined immobilization/ permeabilization process with retained plant cell viability. Complex coacervate capsules consisting of chitosan and alginate or carrageenan proved to be effective biomaterials for entrapment, controlled permeabilization of cells and to allow control of capsule membrane diffusivity. 

Bhakdi, S, Weller U, Walev I et al. (1993) A guide to the use of pore-forming toxins tor controlled permeabilization of cell membranes. In Med Microbiol Immunol 182 167-175. 

Organic solvents like methanol or acetone also can be used to open membranes, but they also act as denaturing fixatives. Solvents are used at -20 C for 10 min, but the use is not recommended. As described in previous sections, denaturing fixatives may cause loss of epitopes in the cells. Other detergent-like agents, such as saponin or digitonin, are used for transient permeabilization of cell cultures, and reversibly insert into and out of plasma membranes next to cholesterol. These agents must be present in all solutions after the fixative and should only be used for electron microscopy immunocytochemistry. 

Johansen, C., Verheul, A., Gram, L., Gill, T., and Ahee, T. 1997. Protamine-induced permeabilization of cell envelopes of gram-positive and gram-nt ative bacteria. Appl Environ. Microbiol 63, 1155-1159. 

There are different techniques to overcome the cell membrane barrier and introduce exogenous impermeable compounds, such as dyes, DNA, proteins, and amino acids into the ceU. Some of the methods include lipofection, fusion of cationic liposome, electroporation, microinjection, optoporation, electroinjection, and biolistics. Electroporation has the advantage of being a noncontact method for transient permeabilization of cells (Olofsson et al., 2003). In contrast to microinjection techniques for single cells and single nuclei (Capecchi, 1980), the electroporation technique can be applied to biological containers of sub-femtoliter volumes, that are less than a few micrometers in diameter. Also, it can be extremely fast and well-timed (Kinosita et al., 1988 Hibino et al., 1991), which is of importance in studying fast-reaction phenomena (Ryttsen et aL, 2000). 

Huang, Y, Rubinsky, B., 1999. Micro-electroporation improving the efficiency and understanding of electrical permeabilization of cells. Biomed. Microdevices 2, 145—150. 

FIGURE ) Permeabilization of cells in suspension. Burkitt lymphoma (Daudi) cells were permeabilized for 0 to 10 min in the presence of trypan blue at 10 cells/ml as described in the text. Permeabilization was determined by trypan blue uptake (% blue = % permeable). Error bars represent standard deviations, three independent experiments (>100 cells counted per assay). 

This chapter discusses protocols for fluorescent labelling of antibodies, optimized fixation, and permeabilization of cells, and describes several immunostain-ing procedures with the aim to quantitatively anafyse the localization of antigens in fixed and living cells. Various aspects of the imaging equipment presently available, and the ways how to most efficiently use it in order to quantify and document results will be discussed. Labelling in living cells by microinjection of fluorescently labelled antibodies and subsequent time-lapse microscopy will also be discussed. 

The field strength controls the extent of peimeabUized membrane, whereas the efficacy of the permeabilization is under the control of the number and the duration of electric pulses [35-37]. This electro-induced permeabilization of cell membrane can be quantified in terms of the flow Fs of molecule S diffusing through the plasma membrane during the post-pulse resealing. In the case of inflow, the molecules can then diffuse freely in the cytoplasm. Pick s law and experimental data allowed to establish that ... 

PEF technology can be used to induce the non-thermal permeabilization of cell membranes and, depending on the treatment intensity (external electric field strength, number and duration of the electric pulses) and cell properties (size, shape, orientation, conductivity), the pore formation may be either permanent or temporary (Zimmermann et al, 1974,1976 Angersbach et al, 2000). 

Carrasco, L., and Lacal, J. C., 1984, Permeabilization of cells during animal virus infection. Pharmacol. Ther., in press. 

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