Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

Summary A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone.This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, ³H-gamma-aminobutyric acid (³H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

Improved methods for using glass coverslips in cell culture and electron microscopy.

For many applications, cells or tissue must be cultured on an optical surface of high quality. For such applications laboratories often prepare "special dishes," which are made by affixing a glass coverslip beneath a hole in a plastic petri dish bottom. In this report, we offer an improved method, using Parafilm as a dry mount adhesive, for the preparation of special dishes, and show that the resulting dish is non-toxic to neurons in culture. The Parafilm bond is stable at 60 degrees C, permitting electron microscopy resins to be poured directly into the dishes and cured. The glass coverslip can be readily removed from the cured resin mechanically. The techniques we describe offer time-saving and reliable improvements for the use of glass coverslips in cell culture and electron microscopy.     

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

Transmission and Scanning Electron Microscopy of Cell Monolayers Grown on Polymethylpentene Coverslips

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized. separated from the coverslip by liquid nitrogen immersion, and sectioned either “en face” or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epithelial macrophages secrete a deactivating factor for superoxide release

The release of superoxide anion (O2-) by inflammatory macrophages, multinucleated giant cells, and epithelioid cells, obtained by the insertion of round glass coverslips into the subcutaneous tissue of mice, was investigated. O2- was shown to be spontaneously released by cells on the surface of glass coverslips implanted up to 7 days, but not by cells obtained 14 or 21 days after coverslip implantation. The former showed increased O2- release when stimulated by phorbol myristate acetate, whereas cells harvested after 14 or 21 days implantation did not. The induction of delayed type hypersensitivity around coverslips implanted for 5 days increased spontaneous O2- release by these cells by 40%. On the other hand, when the same protocol was used with coverslips implanted for 14 days, O2- release was not detected. These results were viewed in regard to the composition of the cell population at each time point. When coverslips were removed after 14 days of implantation and the cells incubated for 30 minutes in vitro, the medium so conditioned inhibited O2- release by cells of 5 day old preparations. This indicates the release by cells on the longer term coverslips of a substance that inhibits O2- production by cells of coverslips implanted for 5 days only. This inhibitory activity could be suppressed by treating the conditioned medium with proteases. The factor was, however, heat stable and exerted its effects even when the test cells were exposed to phorbol myristate acetate.     

Some speculations on the rate of adhesion of cells to coverslips

Suspensions of mouse tumour cells were cultured over glass coverslips, and their rate of adhesion to the coverslips measured. The interaction energies between the cell and coverslip surfaces were computed according to DLVO-theory, on the basis of experimentally determined parameters, for a wide range of values of Hamaker's coefficients. The secondary attractive minimum was computed to occur when the two surfaces were separated by 40–85 Å. It was observed that 50% of the cells adhered to the coverslips after 30 minutes, although it was calculated that they could gravitate to the position of the secondary minima in approximately 5 minutes. We have discussed this delay of 25 minutes in terms of the mechanisms of cell adhesion to the coverslips.     

Evaluation of 40 × 22 mm coverslip area in cervical cancer screening

oommen r., curling m., adams c., barrett e., titmuss e. and widdowson a. (1998) Cytopathology 9, 46–49
Evaluation of 40 ×3 20 mm coverslip area in cervical cancer screening
Nine hundred and twenty-three smears covered by 40 × 22 mm size coverslips were examined inside and outside the coverslip area to determine whether this coverslip size could be responsible for missed dyskaryotic cells in conventional cervical cancer screening. There was no instance when abnormal cells seen outside the coverslip were not also present within the coverslipped area.     

Photoengraving of coverslips and slides to facilitate monitoring of micromanipulated cells or chromosome spreads

A simple photolithographic technique has been developed which can be used to produce microscopic grid patterns on glass coverslips. The grid pattern is first photo-reduced onto film, and the resulting photographic negative is then used as a mask. A glass slide or coverslip, coated with a layer of photoresist, is then exposed to tungsten light through the mask. After developing and etching, the grid pattern is transferred permanently onto glass. This simple and rapid procedure allows one to mass-produce very small, high resolution grids which are useful for monitoring individual microinjected cells or chromosomal spreads under the microscope.     

Effect on germination and post-germination development of Colletotrichum dematium var circinans due to light and dark incubation and coverslip placement

Light regime was not a major factor in germination of spores of Colletotrichum dematium var circinans. Number of germ-tubes and appressoria formed were affected by incubation regime and coverslip placement. Most germ-tubes and appressoria were produced on spores under shimmed coverslips. Light incubation favored germ-tube production and dark incubation favored appressoria production. Predicted time to 100% germination was affected by treatments. Shortest times were predicted for light incubated spores under shimmed coverslips. Longest times were predicted for non-coverslipped spores. A significant number of appressoria were produced sessile on spores. Most sessile appressoria were produced on dark incubated spores. Approximately 56 % of all spores germinated, and approximately 60% of all germ-tubes and appressoria were produced on these spores which were situated closest to coverslip or droplet edges. Placement of germ-tubes on spores was not affected by treatments. However, percentages of germ-tubes, initiated from various areas of spores, were significantly different. This variation appears to be due to factors internal to spores.     

Scyphozoan jellyfish's mesoglea supports attachment, spreading and migration of anthozoans' cells in vitro

Mechanically and enzymatically dissociated cells from five anthozoan species were laid on seven substrates in vitro. Cells were taken from two sea anemones (Aiptasia sp. and Anemonia sulcata), a scleractinian coral (Stylophora pistillata) and two alcyonacean corals (Heteroxenia fuscescence and Nephthea sp). Substrates tested: glass (coverslips), plastic (uncoated tissue culture plates), type IV collagen, gelatin, fibronectin, mesoglea pieces from the scyphozoan jellyfish Rhopilema nomadica and acetic acid extract of jellyfish mesoglea. Except for the mesoglea pieces, cells did not respond to any one of the other substrates, retaining their rounded shape. Following contact with mesoglea pieces, cells attached and spread. Subsequently they migrated into the mesogleal matrix at a rate of 5-10 microm/h during the first 2-5 h. No difference was found between the behavior of cells from the five different cnidarian species.     

Studies of the Numbers and Morphology of the Intracellular Form of Leishmania donovani Grown in Cell Culture*

SYNOPSIS. Macrophages were infected in vivo with the intracellular form of Leishmania donovani (LDs), harvested from the previously saline-stimulated peritoneal cavities of hamsters and explanted into Leighton tubes containing removable coverslips. Serum from either rabbit, chicken, human, calf, hamster or cotton rat blood was used as the 40% component of a Hanks' BSS₆₀ serum₄₀ medium used to maintain these Leighton tube cultures at 37 C. After varying lengths of time coverslips were removed from tubes, stained with Giemsa, and the parasites per infected macrophage, total number of hamster cells and total number of parasites on each coverslip were counted. Maerophages constituted more than 90% of the explanted cells on the coverslips. When cotton rat serum was used as a component of the medium, fibroblastic overgrowth of the coverslips followed. Some similarities and differences in the numbers of macrophages, fibroblasts and parasites were noted with regard to the serum used as part of the medium. Except for cotton rat serum, the serum component of the medium used apparently did not influence, to any great degree, the morphology of either the macrophages or parasites therein. Thus, vacuolarization and granularization of macrophages did not appear to be very distinctly correlated with the time of sampling or the type of serum in the medium used for maintenance nor could any morphologic variations of the LDs be ascribed to these factors. When cotton rat serum, but not any of the other sera, was the serum component of the medium, leptomonads were noted in the overlay fluid of the cultures after 6 days. Under these conditions of cell culture, fibroblasts could not be infected with LDs although macrophages on the same coverslip were heavily parasitized.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Phagocytosis and giant cell formation at 0° C by macrophage (MØ) of Notothenia coriiceps

Macrophages adhered to round glass coverslips that had been implanted into the abdominal cavity of Notothenia coriiceps , collected from Admiralty Bay, King George Island, and removed and incubated in vitro for 4 h at 0° C at various intervals. The macrophages phagocytosed yeast Saccharomyces cerevisiae cells suspended in saline. Bi-nuclear macrophages were first observed after 6 h. The formation of giant cells and their phagocytic activity was observed only in fish which had been injected with 1 ml of Bacillus of Calmette and Guerin (BCG)-suspension 24 h before coverslip implantation and the coverslips removed 15 days after the implantation. Phagocytosis and the formation of giant cells in an Antarctic fish at 0° C is described for the first time.     

A light and electron microscope study of long-term organized cultures of rat dorsal root ganglia

Dorsal root ganglia from fetal rats were explanted on collagen-coated coverslips and carried in Maximow double-coverslip assemblies for periods up to 3 months. These cultured ganglia were studied in the living state, in stained whole mounts, and in sections after OsO₄ fixation and Epon embedment. From the central cluster of nerve cell bodies, neurites emerge to form a rich network of fascicles which often reach the edge of the carrying coverslip. The neurons resemble their in vivo counterparts in nuclear and cytoplasmic content and organization; e.g., they appear as "light" or "dark" cells, depending on the amount of cytoplasmic neurofilaments. Satellite cells form a complete investment around the neuronal soma and are themselves everywhere covered by basement membrane. The neuron-satellite cell boundary is complicated by spinelike processes arising from the neuronal soma. Neuron size, myelinated fiber diameter, and internode length in the cultures do not reach the larger of the values known for ganglion and peripheral nerve in situ (30). Unmyelinated and myelinated nerve fibers and associated Schwann cells and endoneurial and perineurial components are organized into typical fascicles. The relationship of the Schwann cell and its single myelinated fiber or numerous unmyelinated fibers and the properties of myelin, such as lamellar spacing, mesaxons, Schmidt-Lanterman clefts, nodes of Ranvier, and protuberances, mimic the in vivo pattern. It is concluded that cultivation of fetal rat dorsal root ganglia by this technique fosters maturation and long-term maintenance of all the elements that comprise this cellular community in vivo (except vascular components) and, furthermore, allows these various components to relate faithfully to one another to produce an organotypic model of sensory ganglion tissue.     

Rapid communication Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Improved growth of cultured brain microvascular endothelial cells on glass coated with a biological matrix

An improved method for culturing primary rat brain capillary endothelial cells on glass has been developed, using a corneal extracellular matrix coat. Since the collagen-coated plastic attachment surface conventionally used for primary cultures of brain microvascular endothelium gives a high level of background fluorescence in microfluorimetric studies, an alternative attachment surface was tested involving no plastic element. Five substrata combinations were examined and a new combination of glass and corneal endothelial extracellular matrix coat was found to provide excellent cell adhesion, culture growth and purity. Other established substrata combinations tested for comparison, either involved plastic, or used glass with collagen or carbodiimide and collagen coating but the last two gave poor endothelial cell adhesion and growth. Our method using this new attachment surface combination results in stable and pure endothelial cultures, as verified by immunocytochemistry, which are suitable for fluorimetric investigations.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

A simple method for ultrastructural enzyme cytochemistry on small volumes and numbers of isolated cells

Summary A simple technique is described for ultrastructural enzyme cytochemistry using small volumes and numbers of single cells. Small aliquots (1–5 l) of single cells suspended in a glutaraldehyde fixative are incubated on poly-L-lysine-coated glass coverslips. Cells in contact with the coated coverslip become immobilized to that support. All changes of solutions are accomplished without repeated centrifugation.