TECHNICAL NOTE |
Correspondence to: Toyoshi Fujimoto, Dept. of Anatomy and Cell Biology, Gunma Univ. School of Medicine, 3-39-22 Showa-machi, Maebashi 371, Japan.
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Summary |
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We describe a simple quick-freezing method to obtain a large fractured plane of the plasma membrane from monolayer cultured cells. Cells were grown on thin gold foil, inverted on a thin layer of gelatin on thin copper foil, and frozen by a quick press between two gold-plated copper blocks precooled in liquid nitrogen. The frozen cell sandwich was mounted on the cold stage of a freeze-fracture device with the gold side up and was fractured by separating the sandwich with a cold fracture knife. When this technique was applied to confluent monolayer cells, large replicas of the E-face of the upper plasma membrane and the P-face of the lower plasma membrane were obtained. The present metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation. (J Histochem Cytochem 45:595-598, 1997)
Key Words: quick freezing, freeze-fracture, freeze replica, plasma membrane, cultured cells, caveolin, immunocytochemistry
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Introduction |
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Quick-freezing is considered as the best way to preserve cellular ultrastructure for electron microscopy. Since the advent of the "slammer" (
Even with a quick-freezing instrument at hand, it is not easy to obtain a wide area of the freeze-fractured plasma membrane, especially from a monolayer of cultured cells. Because the thickness of a monolayer is usually less than 10 µm, a precise knife advance mechanism is required for freeze-fracturing. Unfortunately, by knife fracturing the fracture plane tends to go through the cytoplasm rather than only the cell surface (
We describe here a simple method to quickly freeze monolayer cultured cells by liquid nitrogen. The method does not require any expensive equipment and, more importantly, produces a large area of the fractured plasma membrane each time.
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Materials and Methods |
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Cells were grown on gold foil (approximate thickness 20-40 µm) cut into a small trapezoid (approximate size: upper side 2 mm; lower side 4 mm; height 6 mm). The trapezoid was intentionally made asymmetrical so that the cell side could be identified easily in subsequent procedures. Gold foil was kept in 1 N HCl and then in ethanol for more than 30 min each, rinsed with distilled water, and autoclaved.
Various cultured cells were dispersed by trypsin-EDTA, seeded on gold foil in plastic dishes (Figure 1A), and maintained in Dulbecco's modified Eagle's medium with the addition of 10% fetal calf serum. As far as we could determine, gold foil is a good substrate for any cell type without any coating. Because cells on foil cannot be observed by light microscopy, the cell density was inferred from surrounding areas in the plastic dish.
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For quick-freezing, a sheet of copper foil (thickness 20 µm) of rectangular shape (approximate size 12 x 20-mm) was prepared. A small amount (5-10 µl) of prewarmed 10% gelatin in PBS was smeared on the copper foil and a piece of gold foil was inverted on the gelatin with the cell side down (Figure 1B). While a corner of copper foil was held with forceps, the sandwich of gold-cell-copper was rapidly pressed between two gold-plated copper blocks that had been precooled in liquid nitrogen. The gold-plated copper blocks were attached to two arms of fine pliers (QF plier; Dosaka EM, Kyoto, Japan) so that the cell sandwich could be grasped in one quick motion. The frozen cell sandwich was kept in liquid nitrogen before further processing.
For freeze-fracturing, the cell sandwich was attached to a specimen table (BB172160-T; Balzers High Vacuum, Balzers, Liechtenstein) by clamping an edge of copper foil by a plate spring; the gold foil was placed on top of the copper foil. The sandwich was transferred to a cold stage of a Bal-zers BAF401 apparatus and a vacuum of 10-7 Torr was obtained. After the temperature was raised to -110C, a cold knife was slowly inserted between the gold and the copper foil. The knife was then moved upwards so that the gold foil was lifted and the frozen cell specimen was fractured (Figure 1C). Platinum/carbon replicas were made as described by the manufacturer (Figure 1D).
The replicas were floated from the specimen in distilled water and transferred to 2.5% sodium dodecyl sulfate (SDS) solution as described (
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Results and Discussion |
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Freezing by clamping has already been applied to tissue pieces (
Replicas obtained by the method were mostly from the fractured plasma membrane. Fracture through the cytoplasm or the extracellular space seldom occurred. Because of the mounting direction of the cell sandwich, fracture planes observable by the present procedure were either the E-face of the upper (dorsal) membrane or the P-face of the lower (ventral) membrane (Figure 1C). The upper and lower membranes are the plasma membranes facing the copper foil and the gold foil, respectively. The proportion of the upper and lower plasma membranes varied for different cell types and probably for different culture conditions, but both membranes can be observed in every replica.
The replicas of the membrane can be used for immunolabeling after treatment with SDS (
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Because this method provides a large planar view of the plasma membrane, the replica obtained can be directly correlated with the result of immunofluorescence microscopy. Figure 3 shows immunofluorescence labeling of caveolin in the lower plasma membrane preparation: In this preparation, the upper plasma membrane and the cytoplasm were removed before immunolabeling. Caveolae are seen as discrete dots and are densely distributed in some areas. Figure 2 apparently corresponds to a region in which caveolae form a dense patch.
The present method was developed based on the one developed by
With a "slammer" or with instruments based on a similar principle, frozen specimens must be cut by a knife to obtain fracture faces. In thin monolayer culture cells, cutting at an exact height is not necessarily easy. However, by lifting off the gold foil, fracture almost always occurred through the cell membrane. The other merit of the metal sandwich method is that large fracture faces of the plasma membrane are revealed each time. Obviously, this metal sandwich can also be frozen by a more sophisticated metal block freezing apparatus, but the metal foil might scratch the mirror surface on contact.
Now that freeze replicas can be a substrate for immunocytochemical labeling, the present method should be useful in revealing the molecular architecture of the plasma membrane in a variety of cultured cells.
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Acknowledgments |
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Supported by a grant-in-aid for Scientific Research (B) (no. 08457001) from the Ministry of Education, Science, Sports, and Culture of the Japanese Government and by a research grant from the Ciba-Geigy Foundation (Japan) for the Promotion of Science.
We thank Ms Natsuko Hatanaka, Yukiko Takahashi, and Fujie Miyata for excellent technical and secretarial assistance.
Received for publication September 4, 1996; accepted November 22, 1996.
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