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.     

Fluoroplastic Coverslips for Long-Term Nerve Tissue Culture

Fluoroplastics (Aclar and Teflon FEP) were tested for their suitability as coverslip material for nerve tissue cultures. Coverslips prepared from these plastics have a higher transparency over the near-ultraviolet, visible, and infrared regions of the spectrum than glass coverslips, possess exceptional physical and chemical stability, and provide service-ability over a wide temperature range ≥ -320 to 390 F. For nerve tissue culture, circular 22 mm coverslips were cut from 5 mil sheets of Aclar 22A and 33C, and Teflon FEP 100A, cleaned in concentrated HNO₃, rinsed, sterilized in 85% ethyl alcohol, dried, and coated with collagen. Peripheral and central nervous system cultures from fetal rats and newborn mice were set up on these as well as on glass coverslips, and carried in Maximow double coverslip assemblies. Over a 2 mo period of cultivation, neuronal maturation, tissue organization and fiber myelination occurred on plastic coverslips in essentially the same manner as in sister cultures maintained on glass coverslips. Cultures fixed and embedded in Epon for electron microscopy directly on Aclar or Teflon 100A coverslips were easily separated from the coverslip. Some difficulty was encountered with retraction of the collagen coat from Teflon 100A coverslips, however, and Aclar 22A coverslips were warped by some of the reagents used in embedding for electron microscopy. Aclar 33C was not adversely affected by these reagents, and provided the most suitable base for collagenization and culture maintenance. A variety of cells grew satisfactorily on either the bare or the collagenized surface of this plastic, and separated easily from it following embedment in Epon.     

Gold labeling of cell monolayers grown on dextran beads

Gold labeling of antigenic sites has become an increasingly useful tool in the study of cultured cell monolayers. If these monolayers are grown on flat substrates, major difficulties in both scanning (SEM) and transmission electron microscopy (TEM) specimen preparation and imaging may result. An alternate surface, that of dextran microcarrier beads, eliminates a majority of these difficulties and facilitates correlative TEM and SEM. The SEM procedure for using backscattered electron imaging requires the use of carbon planchets as the cell growth matrix to eliminate background signals. These planchets are expensive and are not an optimal cell-attachment matrix in that they result in loose and abnormally shaped cells. In contrast, the dextran beads were produced specifically for cell culture and, therefore, provide an excellent surface for growth. The beads have an average diameter of 100 microns, allowing attachment directly to aluminum stubs without signal generation from the aluminum to interfere with the gold signal. With TEM preparation, the monolayer poses the major disadvantage. Specimen preparation for thin sectioning is often preceded by extensive manipulation. In the microcarrier bead system, the beads are directly sectionable, and it is possible to cut five to eight full beads per thin section. This increase in cell surface makes quantification of gold labeling easier and also provides a more representative sampling of the monolayer. The ease of preparation, the decrease in reagents used (via cell pooling), and the ability to use one cell preparation for TEM and SEM make this procedure an ideal technique for gold labeling.     

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.     

A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy

Human leukocytes fixed in suspension were allowed to settle onto poly-L-lysine-coated glass coverslips and prepared for observation with the scanning electron microscope (SEM). The coverslips were dehydrated in ethanol, critical point dried with CO2, and coated with gold-palladium. With the aid of a locator grid, several fields were photographed with light microscopy after the cells had settled onto the poly-L-lysine-coated coverslips and again after completion of the processing before SEM observation. Quantitative comparison of the number of cells present after settling with the number retained for final viewing with the SEM revealed a cell yield approaching 100%. This simple, reproducible, high-yield technique for processing cells fixed in suspension for SEM prevents changes in surface architecture induced by collecting live cells onto various substrates before fixation and also avoids potentially selective cell losses. Such a technique should allow quantitative correlations between SEM and other morphological and functional parameters.     

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.     

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.     

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.     

A "hanging" coverslip method for cell surface iodination of monolayer cultures

A procedure is described by which the use of a 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (iodogen)-coated coverslip to iodinate the cell surface proteins of monolayer cultures has been improved by "hanging" the coverslip at a defined distance from the cells. This method allows gentle manipulation of the cell culture, resulting in retention of high cell viability and in recovery of the cell monolayer with a minimum of mechanical damage. In addition, it allows the safe disposal of the radioactive coverslip upon completion of the reaction. Finally, the labeling is surface specific. The application of this procedure to 3T3 fibroblasts results in labeling of proteins comparable to lactoperoxidase-catalyzed iodinations.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.     

Direct staining and visualization of endothelial monolayers cultured on synthetic polycarbonate filters

Endothelial and epithelial cells cultured on synthetic filter supports have been used to study permeability and transport under various experimental conditions. However, because of the non-transparent nature of these filters, morphological studies using light microscopy are not possible. Presently, investigators circumvent this problem by using cells cultured on glass coverslips, extrapolating morphological data from a system clearly different from that used for functional studies. We describe here a useful technique for direct staining and visualization of cells grown on polycarbonate filter supports, using fluorochrome probes and fluorescence microscopy. We have utilized acridine orange, rhodamine phalloidin, and an anti-vimentin monoclonal antibody to provide information about cell shape, monolayer configuration, and cytoskeletal protein distribution in cultured calf pulmonary artery endothelial cell monolayers. Comparison staining of coverslip and filter preparations revealed a number of clear differences in these parameters. This technique should enable investigators to perform the necessary studies to obtain direct correlations between functional and morphological data.     

Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells

We have developed a method for simultaneous visualization by electron microscopy of both the distribution of cell surface receptors and architectural features of the inner membrane surface, such as clathrin-coated pits. Electron microscope grids were covered with formvar and coated with poly-L-lysine. These grids were then placed on a piece of buffer-impregnated cellulose acetate membrane filter maintained at 4 degrees C on an ice bath. Cells of interest were grown on glass coverslips and incubated with either a ligand-gold or an antibody-gold conjugate specific for the membrane determinant of interest. The coverslip with gold-labeled cells was then overlaid on the grids and pressure was applied. When the grid was removed, large areas of the upper cell surface, which had labeled determinants, remained adherent to the formvar support. With the proper staining, both the gold particles and internal membrane features could be seen at the same time in the electron microscope. This method is rapid, does not require extensive experience with electron microscopic technique, and permits viewing of membrane samples that are large enough to perform quantitative analysis of gold distribution in relation to membrane specializations.     

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.     

Immunocytochemistry: human neural stem cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only approximately 25 microl of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.     

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 of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

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.     

Dynamic molecular combing: a 'low tech' method for genetic mapping and diagnosis

So much of molecular genetics involves invisible samples at the bottom of minuscule tubes, that the attraction of something you can actually see is virtually irresistible. Recent developments in high-resolution fluorescent in situ hybridization (FISH) are both visually appealing and a valuable addition to the repertoire of gene-mapping and diagnostic techniques. The latest of these, dubbed dynamic molecular combing allows precise, high-resolution mapping of markers on cloned or total genomic DNA. In this technique, a coverslip coated with silane is dipped into a DNA solution. After a few minutes' incubation, during which the ends of the DNA molecules bind to the coverslip, a small motorized device is used to withdraw the coverslip vertically, by its edge, at a constant rate of 300 microseconds⁻¹. The force exerted on the DNA molecules by the meniscus stretches all the molecules uniformly in the same direction, and they dry instantly onto the silanized surface as it is exposed to air. The result is a surface covered with a high density of parallel DNA molecules, which can then be probed with fluorescent-labelled markers and visualized under a miscroscope. Because of the constant stretching factor of 2 kb per micrometer of fibre length, no internal control is needed for calibration, unlike other recent fibre-FISH methods. In pilot experiments, the technique has been used successfully to measure microdeletions involving the tuberous sclerosis 2 gene, for fine-mapping and orienting of sets of contiguous cosmid clones (contigs) on a yeast artificial chromosome within its yeast genome, and to measure gaps in a cosmid contig by hybridizing pairs of cosmid probes to combed total human genomic DNA. Dynamic molecular combing will also be useful for a variety of applications in genetic diagnosis, such as mapping the breakpoints where segments of different chromosomes have been interchanged, and analysing sequence rearrangements within genes. Probes as small as 3 kb can be used when mapping cloned DNA; when total genomic DNA is the template, however, the probe needs to be at least 5 kb to enable the signal to be distinguished from background. The length of DNA that can be visualized in any one field of view, approximately 400-600 kb, is determined by the characteristics of the camera used to record the image and the objective on the microscope. The new equipment needed is simple and the protocol very straightforward, which should put it within the reach of most genetics laboratories. The motorized device used to withdraw the coverslip from the DNA solution at the required speed of 300 micrometer s⁻¹ will be available commercially from early 1998 (contact the authors of the Science paper for details). Alternatively, such a device could be readily constructed by most departmental workshops. The silane-coated coverslips are best prepared in the gaseous phase, especially for combing total genomic DNA, when a homogeneous surface is essential. The researchers in Paris and London who developed the technique are, therefore, also considering making the coverslips commercially available.     

Piezoelectric effect stimulates the rearrangement of chondrogenic cells and alters ciliary orientation via atypical PKCζ

Therapeutic ultrasound was administered to patients suffering from bone fracture with FDA approval. Bone and cartilage are piezoelectric materials. To investigate the effects of piezoelectricity on the cells of chondrogenic lineage, we applied ultrasound stimulation on an AT-cut quartz coverslip to generate electric field fluctuations. The bone-marrow-derived mesenchymal stem cells (BMMSC) and primary chondrocytes were cultured on either glass or quartz coverslips for ultrasound stimulation. The cells were immunofluorescent-labeled for the assessment of cell arrangement and ciliary orientation. Ultrasound and piezoelectricity both stimulate cell migration and disrupt ciliary orientation induced by directional migration. In particular, piezoelectric effects on cell rearrangement can be abolished by the inhibitor specifically targeting atypical Protein kinase C zeta (PKCζ). Our findings shed light on the possibility of cellular modulation by using piezoelectric manipulation.     

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.