Adhesion of suspension cells on a coverslip in serum-free conditions

Here, we present a rapid and damage-free fixation protocol for human cells cultured in suspension. Our results demonstrated that serum-free incubation of myeloid suspension cell lines HL-60, U937, and THP-1 for 10 min resulted in cell adhesion to coverslips, allowing simple and efficient fixation for microscopy. The fixed cells exhibited an intact morphology and were suitable for immunostaining. Such simplicity and cost effectiveness have not been achieved by any previously established fixation technique, and our newly developed method provides an additional fixation technique for researchers working with suspension cells.     

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.     

Changes in plasma-membrane-associated filaments during endocytosis and exocytosis in polymorphonuclear leukocytes

We examined the filaments associated with the cytoplasmic surface of the plasma membrane in rabbit exudate PMNs during phagocytosis of particles, or during “frustrated phagocytosis” with exocytosis of storage granules. Cells were plated onto yeast particles glued to coverslips with polylysine or onto coverslips coated with sheets of heat-agglutinated IgG. After periods ranging from 1 to 15 min, we disrupted the cells by a jet of salt solution and exposed their inner membranes. These broken cells were fixed immediately and processed for SEM. Whole cells were also prepared for SEM or TEM. At the site of PMN adherence to an opsonized yeast particle, a network of globular centers and thin, branched filaments appears on the cytoplasmic surface of the plasma membrane, while the outstretching lamellipodia contain a mesh of such filaments but no globular centers. Within 1 to 2 minutes, these structures disappear from the invaginating portion of the developing vacuole, and the cell's storage granules fuse with the barren membrane regions. These activities occur in rapid sequence over the vacuolar membrane after the first contact, until the phagocytosed particle is wholly encircled by a smooth, loose membrane, separated from the cell surface. A comparable filament pattern or complex was seen during “frustrated phagocytosis” on IgG sheets. At times between 1 and 5 min after plating, the cytoplasmic surfaces of these adherent membranes contain denuded central regions and peripheral nets of globular centers with radiating, thin, branched filaments. Granules apparently fuse with the bare areas. Thus we have obtained evidence of filament association with the plasma membrane at sites of adherence (to phagocytosable or nonphagocytosable surfaces) and have traced the subsequent disappearance of the filaments with degranulation.     

Scanning force microscopy reveals ellipsoid shape of chicken erythrocyte nucleosomes.

Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30.     

A Technique for Continuous Observation of the Infection Process in Cucumber Anthracnose

A new light microscope preparation technique for high magnification observation of living plant tissue and fungal penetration is described. Agar immersion is used in differential interference contrast microscopy (DIC) instead of coverslips (lens 40) or instead of coverslips and oil (lens 100).
This technique is suitable for
(a) longtime observation of living tissue, because the tissue to be observed remains on the plant, and for
(b) thick and uneven samples, as no coverslips are required.
With this technique it was possible to observe the dynamics of penetration of Colletotrichum lagenarium into epideral cells of cucumber cotyledons for 72 hours.
A time lapse film using this technique is in preparation.     

Confocal microscopy of surface- and cytoplasmically-labelled bacteria immobilized by APS-centrifugation

Cells of Escherichia coli and Staphylococcus aureus were immobilized by centrifugation onto glass coverslips treated with 3-aminopropyltriethoxysilane (APS-centrifugation), labelled with fluorescein isothiocyanate (FITC), fixed with acetone and counterstained with propidium iodine (PI). Confocal microscopy of these preparations allowed resolution of the expected surface (FITC) and cytoplasmic (PI) labels and serial optical sections through cells were readily obtained. The APS-centrifugation technique provides immobilized bacteria in a form which can take full advantage of confocal microscopy.     

Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane- associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.     

Fixed bed porous glass sphere (porosphere) bioreactors for animal cells

Process intensity of fixed bed glass sphere culture systems is increased considerably by replacing solid glass spheres with open pore glass spheres. This technique demonstrates the possibility of having a system capable of both volumetric and cell density scale up and being suitable for substrate attached and suspension cells. The yields achieved for a number of attached cell lines (approximately 10⁷/ml) demonstrate an increase approaching one order of magnitude over solid glass spheres (approximately 10⁶/ml). Also suspension cells were successfully entrapped in the open pore structure with similar yields.     

Transmission Electron Microscopy of Tissue Culture Preparations on Polystyrene Coverslips

Polystyrene coverslips have been utilized in preparing tissue cultures for transmission electron microscopy. A method is described for processing the coverslips, including the marking of selected groups of cells with epoxy-carbon mixture. The epoxy-carbon markings aid in locating the cells during sectioning and the method can be used for small or large numbers of cells. In addition, the methodology allows high power phase contrast photography before and after fixation.     

A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES

Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.     

Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase

Background. Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori‐induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase. Materials and Methods. To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM. Results. We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours. Conclusions. Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori‐induced gastric disorders by SEM.     

An anchorage-dependent locus in the cell cycle for the growth of 3T3 cells

Mouse fibroblasts, 3T3 cells, require a solid surface for continuous growth, but when 3T3 cells, during their exponential phase in Petri dishes, were transferred to a suspension culture, the number of cells roughly doubled by 30 h. During the suspension culture the number of pairing cells (c₂) increased, but that of the single cells decreased. When cells synchronized at mitosis or at the G1-S boundary were transferred to the suspension culture, the number of pairing cells peaked at 30 min and at 10 h, respectively. DNA synthesis began immediately after the cells, which were cultured for 16 h in the suspension, had settled onto the surface of the Petri dishes. When cells in a confluent culture were arrested at an early G1 period and were suspended, the number of pairing cells did not increase. These results indicate that the most important locus for anchorage growth seems to be at a late G1 period of the cell cycle.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

A Colchicine, Hypotonic Citrate, Air Drying Sequence for Foetal Mammalian Chromosomes

Mice 14 days pregnant were given 0.3 ml of 0.025% Colcemid (Demecolcine, Ciba) and killed after 1 hr. The livers of the foetuses were removed and broken up in 0.1% Colcemid in phosphate-buffered 0.85% NaCl and left 1.5 hr. The cell suspension was then centrifuged, resuspended in 1% sodium citrate for 20 min, centrifuged and the cells fixed in acetic-alcohol (1:3) for 30 min at 4°C. The cells were then resuspended (twice) in 45% acetic acid and dropped onto warm slides. After drying, the cells were stained for 30 min with lactic-acetic-orcein and examined under oil-immersion without a cover slip. Good numbers of well-spread mitotic figures were obtained.     

High resolution scanning electron microscopy of frog sartorius muscle

A field emission-type scanning electron microscope (SEM) was used to study the three-dimensional ultrastructure of frog sartorius muscles. Various preparative procedures were tested to seeks better specimen preparation for high resolution SEM observation. Procedures should be chosen depending on the information desired. The cell surface and intracellular organization of muscle fibers were best visualized when the tissues were fixed with tannic acid-OsO4 and torn after critical point drying. The basal lamina appeared as a continuous felt-like layer, onto which fine filamentous materials adhered. The true outer surface of the sarcolemma was not seen, whereas the true inner surface was occasionally exposed and exhibited numerous caveolae, membraneous fragments and fine filaments attached to its surface. In freeze-fractured and dried tissues, the cleaved sarcolemma showed numerous apertures of caveolae and T-system tubules. Inside the cell, the myofibrils showed a typical branding pattern of the sarcomere. Thick myofilaments were regularly beaded except for the pseudo-H-zone. Around the myofibrils the sarcoplasmic reticulum and T-system were also clearly observed. The results are discussed with special reference to the usefulness and limitation of the high resolution SEM in studying the ultrastructure of cells and tissues.     

A new method for the cytochemical staining of cells immobilized in agarose

Summary A quick, simple and inexpensive technique utilizing an isotonic matrix of semi-solid agarose for immobilizing channel catfish (Ictalurus punctatus) peripheral blood leucocytes onto microscope slides before cytochemical staining is described. The technique was demonstrated to be superior to the more conventional means of immobilizing cells for procedures requiring either fixed (Sudan Black B and nonspecific esterase stains) or viable (Nitro-Blue Tetrazolium stain) cells. Although the agarose immobilization technique was developed with channel catfish leucocytes, its utility with other fragile cell populations seems likely.     

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.     

Control of peripheral glial cell proliferation: a comparison of the division rates of enteric glia and Schwann cells and their response to mitogens

The enteric nervous system comprises neurons and a relatively homogeneous population of glial cells, which differ considerably from those found in other parts of the peripheral nervous system and resemble more closely astrocytes from the central nervous system. It provides a simple model system for the study of neuron/glial interactions and glial cell development. In this study the proliferation rates of purified populations of enteric glia and Schwann cells and their response to several mitogens in vitro were compared. Enteric glial cells divided at a much higher rate than Schwann cells in both serum-containing and serum-free media. This difference in their basal proliferation rates was the major difference seen between the two cell types. Both cell populations were stimulated to divide by fibroblast growth factor and glial growth factor but not by epidermal growth factor. Enteric glial cells and Schwann cells proliferated at a greater rate on a basement membrane-like extracellular matrix produced by corneal endothelial cells, laminin, and fibronectin than on poly-L-lysine-coated glass coverslips. The magnitude of stimulation was greater for Schwann cells, presumably due to their lower basal division rates. Like Schwann cells, enteric glial cells were stimulated to divide by two agents which elevate intracellular cAMP, cholera toxin, and dibutyryl cAMP.