Permeabilization with Saponin

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). 

The use of saponin, a plant glycoside, for permeabilization was introduced by Endo and coworkers (1977). Saponin removes the surface membrane without impairment of the functions of SR. The plasma membrane, unlike that of triton skinned smooth muscle (Spedding, 1983), appears fairly intact in electron microscopic pictures of cross sections of permeabilized cells. However, when patches of isolated membranes were viewed face on in homogenates of permeabilized cells, numerous 70- to 80-A holes were visible (Kargacin and Lay, 1987). 

Fibers treated in the described way respond rapidly to increasing Ca + concentrations. Threshold concentrations are around 2 x 10 7 M Ca + and maximum contraction was observed at 10 (jlM Ca +. Maximal force is comparable to or even larger than that observed before saponin treatment. This was taken as a criterion for complete skinning (Endo et ah, 1982). As in triton skinned fibers, tension development depends on the presence of ATP (Endo et ah, 1982). 

Except for one study (Haeusler et ah, 1981), no response to agonists was observed after saponin per-meabilization. Haeusler modified the saponin per-meabilization using high concentrations of saponin (500 xg/ml) for a very brief period (5-6 min). Mesenteric arteries skinned in this way responded to noradrenaline with a contraction that was attributed to release of Ca2+ from internal stores. Saponin-treated smooth muscle, however, responds to GTP7S, a poorly hydrolyzable GTP analog, which permanently activates G proteins (Fujiwara et ah, 1989 Hirata et ah, 

This suggests that the disruption of phar-macomechanical coupling in saponin-treated smooth muscle is due to disruption of the coupling between surface-membrane receptors and their G proteins while the G protein effector cascade appears to be functional. 

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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.

Smooth muscle preparations permeabilized with saponin, p-escin, or a-toxin have significantly contributed to the understanding of Ca2+ release from intra-... 

Incubate coverslips a second time with a mouse anti-Francisella EPS antibody dilution for 40 min at room temperature in permeabilization/blocking buffer. Because cells are fully permeabilized with saponin, this step allows the antibody to reach all intracellular bacteria. 

Fig. 8.16. Cell cycle analysis of CD8-positive and CD8-negative cells. Lymphocytes were cultured with phytohemagglutinin, stained with FITC anti-CD8 monoclonal antibody, treated with saponin to permeabilize the outer membrane, and then stained with propidium iodide (PI) and RNase. Cells provided by Ian Brotherick.

Escin and a-toxin have attracted a lot of interest since permeabilization with either one of them appears not to interfere with the receptor effector coupling. This allows the study of the intracellular signaling cascades involved in pharmacomechanical coupling. (3-Escin is a saponin ester and a-toxin is a bacterial toxin from Staphylococcus aureus. They differ by the size of fhe holes they produce in the plasma membrane. 

FIGURE 22-2 ATP-dependent uptake of calcium into endoplasmic reticulum and mitochondria as a function of extraorganellar Ca2+ concentration. As [Ca2+] j was raised, ATP-dependent Ca2+ uptake into the endoplasmic reticulum (ER) and mitochondria] pools increased. Data for ER uptake were determined as the amount of Ca2+ [45Ca2+] taken up by saponin-permeabilized hepatocytes after addition of ATP and in the presence of mitochondrial inhibitors. The curve for mitochondria] uptake was obtained by subtracting the ATP-dependent uptake in the presence of these inhibitors from that in the absence of inhibitors. For additional details consult Burgess et al. [31]. Stylized data taken from results originally reported in Burgess etal. [31]. (Redrawn and modified from reference [4] with permission of Landes Bioscience.)... 

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. 

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

Pellet the cells and wash twice with 1 mL FACS-wash A, vortexing gently and centrifuging the cells at 1100 rpm with each wash, then fix and permeabilize the cells in Permeafix (1 mL) for 40 min at room temperature (see Subheading 3.3.1. for saponin permeabilization). 

The permeabilization protocol with (3-escin is similar to that applied for saponin skinning (Kobayashi et ah, 1989). The pores are quite large, allowing even antibodies to permeate (lizuka et ah, 1994). In these preparations, the pharmacomechanical coupling is functionally intact as judged from the agonist-induced release of Ca + from intracellular stores and increase in Ca + sensitivity of the myofilaments (Kobayashi et ah, 1989). One criterion for complete permeabilization again is the amplitude of maximal Ca +-activated... 

In general, the stores are loaded with Ca + buffered with millimolar concentrations of EGTA (Endo et al., 1977 Saida and Nonomura, 1978 Itoh et al., 1982a,b Saida, 1982). The stores are fully loaded within 3 to 5 min at 1 (jlM Ca2+ (Saida, 1982), whereby the Ca2+ uptake depends on the Ca + concentration (Saida, 1982 Yamamoto and van Breemen, 1986). At Ca + concentrations greater than 1 jlM, a Ca +-induced Ca release was observed (Itoh et al., 1981 Saida, 1982). The deposition of Ca2+ in the SR of saponin-permeabilized smooth muscle was demonstrated by electron probe X-ray microanalysis (Kowasaki et al.,... 

Incubate cells for 2 min at room tanperature in 0.5 mL/well saponin solution (140 mM potassinm glutamate-HCl, pH 6.8, with 1 mg/mL ATP and 0.1 mg/mL saponin) to permeabilize the cell monbranes. Optionally, 50 xM PMSF can be added to the saponin solution to irreversibly inhibit amidase enzymes if endo-cannabinoids are to be added or their endogenous activity is to be studied. 

To allow antibody access to intracellular structures and simultaneously saturate antibody non-specific binding sites, retrieve each coverslip from its well, drain excess liquid onto a Kimwipe, and invert it (cells facing down) on top of a 50 pL drop of permeabilization/blocking buffer. Incubate for 30 min to 1 h at room temperature. Saponin is the mildest detergent for sample permeabilization and is reversible. Hence, it is kept present in antibody dilutions subsequently used. Alternatively, some antigens require extraction with 0.1% Triton X-100 in PBS for 5 min at room temperature, which irreversibly permeabilizes the cells see Note 8). 

Cells are next blocked and permeabilized by incubating for 30 min with PBS containing 10% fetal bovine serum and 0.05% saponin (blocking buffer). We find manipulation of the coverslips is easiest by performing all labeling steps on parafilm on the bench top. Covershps are then inverted onto 50-100 yX drops of blocking buffer into which primary antibody has been diluted and incubated for 30 min. 

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