Saponin, Permeabilization

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). 

Paraformaldehyde fixes by crosslinking surface proteins. Ethanol and methanol work through precipitation of membrane components. Triton X-100 and Tween-20 permeabilize membranes. Other permeabilizers are saponin (0.1-10% [v/v] in PBSG), L-lysophosphatidylcholine, and n-octyl-P-D-glucopyranoside (1-10 pg/mL in water). 

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.

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.

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. 

PBS containing 0.2% fish-skin gelatin (PBS-gelatin). For permeabilized cells (see Subheading 3.), 0.05% Saponin is added (PBS-gelatin-Sap). 

Permeabilizing agent, e.g., Permeafix (Ortho Diagnostics, UK), saponin. 

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). 

Key words Permeabilization, Organic solvents, Detergents, Saponin, Triton X-100, Tween 20... 

Permeabilizers Triton-XlOO, Tween 20, saponin, 1-lysophos-phatidylcholine, n -octyl-beta-d-glucopyranoside. 

Authi KS, Rao GHR, Evenden BJ, Crawford N (1988) Action of Guanosine 5 -(beta-thio)Diphosphate on Thrombin-Induced Activation and Calcium Mobilization in Saponin-Permeabilized and Intact Human Platelets. Biochem J 255 885... 

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. 

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). 

---