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PubMed1996

Exposure to asbestos fibres during gasket removal.

In the chemical industry, asbestos gaskets have been used extensively to prevent leakage between solid surfaces. The purpose of this study was to assess the potential personal exposure to asbestos fibres during gasket removal and thus to determine what is reasonably required in terms of protective regime and work method. Two groups were studied: group A, who only removed gaskets if they could be removed easily and without breaking; and group B, who removed gaskets which were left by the first group. For both groups, the gasket was first made wet before removal. The samples were analysed using phase-contrast microscopy and transmission electron microscopy. The average exposure to fibres for group A, averaged over the work period, was 0.04-0.242 fibres ml-1 as determined by phase-contrast microscopy. On further analysis with transmission electron microscopy it was found that only four of the 11 samples contained asbestos in very low concentrations and that most of the fibres, which were identified using phase-contrast microscopy, probably originated from the glass fibre lagging around the pipes. For group B the average fibre concentration, averaged over the work period, as determined by phase-contrast microscopy, ranged between below the detection limit and 0.02 fibres ml-1. The subsequent transmission electron microscopy analysis shows that the exposure to asbestos fibres ranged between below the limit of detection to 0.004 fibres ml-1. This study shows that the exposure to asbestos fibres during gasket removal activities was well within the 8-h average exposure limit of 0.3 fibres ml-1.

Asbestos↗

PubMed2001

Phase separation in gelatin/maltodextrin and gelatin/maltodextrin/gum arabic mixtures studied using small-angle light scattering, turbidity, and microscopy.

The kinetics of phase separation were observed in the gelatin/maltodextrin and gelatin/maltodextrin/gum arabic systems, where gum arabic was added as a minority component, using small-angle light scattering, turbidity measurement, and confocal scanning laser microscopy. Phase separation occurred by spinodal decomposition for quenches both above and below the temperature at which gelatin gelled. Coarsening of the phase-separated microstructure was hindered by gelation, and a hydrodynamic mechanism, observed when the gelatin remained in the liquid state, was suppressed. Gum arabic, containing both polysaccharide and polypeptide components, was hypothesized to be potentially interfacially active in the gelatin/maltodextrin system, in analogy with synthetic block copolymer compatibilizers in demixed synthetic polymer systems. The hypothesis was experimentally refuted under the chosen experimental conditions, as no evidence was found to suggest that it altered the phase separation behavior.

Biopolymers↗

PubMed1976

In vitro accumulation of mineral components by invertebrate cartilage.

In vitro mineralization of the gill cartilage of Limulus (horse-shoe crab) has been reported previously (Eilberg et al. 1975). The present study demonstrates that cranial cartilage of Loligo (squid) and odontophore cartilage of Busycon (marine snail) also mineralize in vitro in hydroxyapatite-metastable media, but not in carbonate-metastable media. In all three of these cartilages, light phase-contrast microscopy revealed that the mineral phase occured in the form of spherical or ovoid granules ranging between 2 and 9 mum in diameter. During mineralization, the granules appeared successively in the perichondrium, in the matrix, and finally, within chondrocytes. Mineralization occurred more rapidly in Eusycon odontophore cartilage which has a significantly higher content of phosphatidyl serine than in Limulus gill or Loligo head cartilages. In all tissues the mineralization process is related to temperature, taking place most actively at 37 degrees C and only weakly at 50 degrees C.

Animals↗

PubMed1996

Metabolites of 5-fluorouracil, alpha-fluoro-beta-alanine and fluoroacetic acid, directly injure myelinated fibers in tissue culture.

The neurotoxicity of two 5-fluorouracil (5-FU) derivatives, tegafur (FT) and carmofur (HCFU), which selectively induce leukoencephalopathy involving the cerebral white matter in humans and vacuolation of myelinated fibers in dogs and cats, was examined in vitro. The common metabolites of these drugs, alpha-fluoro-beta-alanine (FBAL) and fluoroacetic acid (FA), were added to the medium of cultured murine cerebellar myelinated fibers. On day 1 of exposure to 7 microM FBAL and FA, which corresponds to their blood concentrations 2 h after oral administration of 10 mg.kg-1 HCFU to dogs that induced central nervous system vacuolation after 30 days, partial splits of the myelinic intraperiod line were observed by electron microscopy. On days 4-7, phase contrast microscopy revealed spindle-shaped swelling and granulation of myelin and electron microscopy demonstrated prominent dissociation of the myelinic intraperiod line with monolocular and multilocular vacuolation. More severe changes, such as myelin loss, were found in cultures exposed to a higher concentration (70 microM) of FBAL and FA, but no remarkable neuronal, astrocytic or oligodendrocytic changes occurred. Quantitative evaluation of myelin injury by electron microscopy revealed significant toxicity of FBAL and FA, at concentrations of 7 and 70 microM, on day 4. However, groups treated with 0.7 microM FBAL and FA, 5-FU (7 microM) and controls exposed to beta-alanine and acetic acid concentrations of 0.7, 7 and 70 microM showed no marked injury. We concluded that these anticancer drug metabolites injure myelin fibers directly, resulting in vacuolation due to myelin splitting and destruction.

Animals↗

PubMed1959

The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation.

A previous electron microscopic study of the cat testis revealed that spermatids derived from the same spermatogonium are joined together by intercellular bridges. The present paper records the observation of similar connections between spermatocytes and between spermatids in Hydra, fruit-fly, opossum, pigeon, rat, hamster, guinea pig, rabbit, monkey, and man. In view of these findings, it is considered likely that a syncytial relationship within groups of developing male germ cells is of general occurrence and is probably responsible for their synchronous differentiation. When clusters of spermatids, freshly isolated from the germinal epithelium are observed by phase contrast microscopy, the constrictions between the cellular units of the syncytium disappear and the whole group coalesces into a spherical multinucleate mass. The significance of this observation in relation to the occurrence of abnormal spermatozoa in semen and the prevalence of multinucleate giant cells in pathological testes is discussed. In the ectoderm of Hydra, the clusters of cnidoblasts that arise from proliferation of interstitial cells are also connected by intercellular bridges. The development of nematocysts within these groups of conjoined cells is precisely synchronized. Both in the testis of vertebrates and the ectoderm of Hydra, a syncytium results from incomplete cytokinesis in the proliferation of relatively undifferentiated cells. The intercellular bridges between daughter cells are formed when the cleavage furrow encounters the spindle remnant and is arrested by it. The subsequent dissolution of the spindle filaments establishes free communication between the cells. The discovery of intercellular bridges in the two unrelated tissues discussed here suggests that a similar syncytial relationship may be found elsewhere in nature where groups of cells of common origin differentiate synchronously.

Animals↗

PubMed1987

Binding specificities of purified porcine brain alpha- and beta-tubulin subunits and of microtubule-associated proteins 1 and 2 examined by electron microscopy and solid-phase binding assays.

In this study, the molecular interaction of separated alpha- and beta-tubulin with purified microtubule-associated protein 1 (MAP 1) and MAP 2 was studied using electron microscopy and solid-phase binding assays with 125I-radiolabeled proteins. Electron microscopy of proteins recovered from sodium dodecyl sulfate polyacrylamide gels and subsequently incubated in various combinations under conditions promoting tubulin polymer formation revealed that both subunits have binding sites for MAP 1 as well as MAP 2. Overlays of nitrocellulose-transblotted MAPs with electrophoretically separated tubulin subunits eluted from gels confirmed these results. In overlays of nitrocellulose-immobilized tubulin subunits with gel-eluted MAP 2, self-association of MAP 2, but no binding to tubulin was detected. However, overlays with MAP 1 and MAP 2 purified under nondenaturing conditions revealed binding of both MAPs to beta-tubulin. In addition, these experiments demonstrated binding of both MAPs to MAP 2 and to the neurofilament proteins NF 70, NF 150 and NF 200. It is concluded that both alpha- and beta-tubulin possess binding sites for MAP 1 as well as MAP 2, but that the accessibility and/or binding affinity of these sites are strongly dependent on the tertiary structure of proteins. The demonstrated in vitro binding of MAP 1 and MAP 2 to all three neurofilament proteins as well as to MAP 2 confirms their presumed role as cytoskeletal linking proteins.

Animals↗

PubMed1975

A new miniature hydrostatic pressure chamber for microscopy. Strain-free optical glass windows facilitate phase-contrast and polarized-light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature.

This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase-contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardation (BR) og 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20 times UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40 times phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chambers, as well as methods for shifting and controlling the temperature within the chamber, are included.

Atmosphere Exposure Chambers↗

PubMed1995

Cryptococcus neoformans survive and replicate in human microglia.

BACKGROUND: Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level. EXPERIMENTAL DESIGN: To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag. RESULTS: Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP. CONCLUSION: The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.

Animals↗

PubMed1997

Light and scanning electron microscopy of rabbit lens capsules with intraocular lenses.

PURPOSE: To examine postoperative changes in the lens capsules of rabbit eyes after phacoemulsification and aspiration of the crystalline lens and implantation of posterior chamber intraocular lenses (IOLs) using light and scanning electron microscopy. SETTING: Research Laboratory, Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: The crystalline lens was emulsified and aspirated and an IOL implanted in the capsular bag or ciliary sulcus of each eye in adult albino rabbits under general anesthesia. Animals were killed after 4 weeks, and the lens capsules were removed. The specimens were observed under phase-contrast microscopy and processed for light and scanning electron microscopy. RESULTS: Phase-contrast microscopy revealed presumed lens epithelial cells (LECs) on the central posterior capsules in association with regenerating lenticular fibers and Elschnig pearls in the peripheral capsules. Scanning electron microscopy showed the accumulation of fibrous extracellular matrix on the surface of the posterior capsule in eyes in which the IOL was implanted in the ciliary sulcus. Deposition of packed material attached to the surface of IOLs and of Soemmering's ring were observed in eyes with in-the-bag IOL fixation. At a higher magnification, a parallel arrangement of lenticular fibers was seen in the regenerated lens structure on posterior capsules. An identical structure was observed under light microscopy. Outgrowth of presumed LECs from residual anterior lens capsules and adhesion of macrophages and giant cells were observed on the IOL surface. CONCLUSION: Two types of postoperative changes were observed in lens capsules after implantation of IOLs: accumulation of fibrous extracellular matrix and newly formed lenticular fibers. These changes are attributed to the proliferation of LECs and can induce posterior capsule opacification after IOL implantation.

Animals↗

PubMed2006

Bitumen morphologies by phase-detection atomic force microscopy.

Summary Bitumen is a complex mixture of hydrocarbons for which microstructural knowledge is incomplete. In an effort to detail this microstructure, 13 bitumens were analysed by phase-detection atomic force microscopy. Based on morphology, the bitumens could be classified into three distinct groups. One group showed fine domains down to 0.1 microm, another showed domains of about 1 microm, and a third group showed up to four different domains or phases of different sizes and shapes. No correlation was found between the atomic force microscopy morphology and the composition based on asphaltenes, polar aromatics, naphthene aromatics and saturates. A high correlation was found between the area of the 'bee-like' structures and the vanadium and nickel content in bitumen, and between the atomic force microscopy groups and the average size of molecular planes made of fused aromatics. The morphology and the molecular arrangements in bitumen thus appear to be partly governed by the molecular planes and the polarity defined by metallic cations.

Journal Article↗

PubMed2003

Human lens epithelial cell damage and stimulation of their secretion of chemical mediators by benzalkonium chloride rather than latanoprost and timolol.

OBJECTIVE: To investigate the effects of latanoprost, timolol maleate, and benzalkonium chloride on cell damage and induction of the secretion of chemical mediators of stress and wound healing by human lens epithelial cells in culture. METHODS: Cells from a human lens epithelial cell line (SRA01/04) were cultured in Dulbecco minimum essential medium supplemented with 5% fetal bovine serum. The amounts of latanoprost (50 micro g/mL), timolol maleate (5 mg/mL), or benzalkonium chloride (200 micro g/mL) used in eyedrops, and x10 to x1000 dilutions thereof, were added to the medium. After 7 days' culture, cell morphological changes were assessed using phase-contrast microscopy, and cell-free culture supernatants were collected for prostaglandin E2 (PGE2), interleukin 1alpha (IL-1alpha), and interleukin 6 (IL-6) iodine I 125 radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent enzyme immunoassay, respectively. RESULTS: All cells that were cultured with the concentrations of latanoprost, timolol, or benzalkonium chloride used in eyedrops detached from the culture dish and died within 3 days. At a x10 dilution of latanoprost or timolol or a x100 dilution of benzalkonium chloride, no proliferation or elongation of the cells was observed. Secretions of PGE2, IL-1alpha, and IL-6 at x10 dilutions of latanoprost or timolol were 3 to 77 times higher than in controls, whereas they were 190 to 305 times higher at a x180 dilution of benzalkonium chloride. The amounts of these soluble mediators in culture supernatants depended on the dose of latanoprost, timolol, or benzalkonium chloride added. CONCLUSION: Our results indicate that benzalkonium chloride, used as the preservative in eyedrops containing latanoprost or timolol, is the agent most damaging to lens epithelial cells and most strongly stimulates the expression of soluble chemical mediators in these cells.

Antihypertensive Agents↗

PubMed1996

Radiotherapy as a cisplatin-sensitizer in a resistant ovarian carcinoma cell line.

BACKGROUND: Stage III ovarian carcinoma has shown resistance to adjuvant chemotherapy following surgical cytoreduction. With recurrence of ovarian carcinoma, cell lines may develop resistance to previously used chemotherapy. This contributes to the fact that survival rates for patients with ovarian carcinoma have not been dramatically improved in decades. The objective of this study is to evaluate radiotherapy as a cisplatin-sensitizer in a cisplatin-resistant ovarian carcinoma cell line. METHODS: In vitro OVCAR-3 human ovarian carcinoma cells were irradiated with external beam radiation (XRT) at doses of 500, 1,500, and 4,500 centigray (cGy) in a single fractionation. Twelve hours after XRT, cells were treated with a dose of cisplatin for 2 hours (0, 1, 3, 9, and 90 micrograms/mL). Cell attachment was determined by cell counts using a hemocytometer under phase-contrast microscopy. Analysis of variance followed by the Student Newman Keuls Test were used for statistical analysis. RESULTS: Dose-response curves demonstrate the results of this study as follows: (1) XRT has a significant direct effect on cell attachment of OVCAR-3 cells in a dose-response relationship. (2) cisplatin has no effect on cell attachment in the absence of XRT. (3) When cells are exposed to XRT, cisplatin demonstrates a dose-response effect on cell attachment with a dose of XRT as low as 500 Gy. CONCLUSIONS: This in vitro study suggests that XRT sensitizes cisplatin-resistant OVCAR-3 to cisplatin. This occurred with doses of radiation low enough to suggest a potential clinical role in treating resistant ovarian carcinoma.

Analysis of Variance↗

PubMed2002

Platelet spherocytosis: a new bleeding disorder.

The present study evaluated a child with thrombocytopenia and a rare congenital bleeding disorder associated with platelet spherocytosis. Differential interference phase contrast microscopy (DIC) revealed that his platelets were spherical in form. Examination of thin sections in the electron microscope showed that his platelets were nearly devoid of microtubules (MT) and microtubule coils (MTC) that support the discoid shape in 100% of normal platelets. Immunofluorescence with a monoclonal antibody to tubulin, the precursor protein of MT, revealed bright rings in normal cells and diffuse fluorescence of patient platelets. The brightness of the fluorescence emitted by patient platelets was comparable to the diffuse fluorescence of normal platelets after chilling to dissolve intact MT, suggesting normal and patient cells contained comparable amounts of tubulin. Exposure of patient platelets to Taxol, an agent that stabilizes and induces MT, caused MT formation in 82% of patient platelets and MTC development in 11%, resulting in their conversion to discs. Glycoproteins GPIIb/IIIa and GPIb were present on patient platelets, and the cells contained normal numbers of dense bodies, ruling out storage pool disease. The patient's platelets adhered to and spread normally on glass but failed to undergo rapid, irreversible aggregation when stirred with agents that produced a complete response in normal discoid platelets, even when the patient's platelets were concentrated. The poor response of spherical platelets was associated with failure to become irregular and extend long filopodia. Thus, the spherical shape of patient platelets may contribute to the thrombocytopenia and to the clinical bleeding symptoms.

Adolescent↗

PubMed1989

Kinetic analysis of thymocyte attachment to thymus stromal cells in culture by using phase-contrast and scanning electron microscopy.

Direct cellular contact between thymocytes and thymus stromal cells within the thymus appears to contribute to the maturation of thymocytes. Thymocyte-stromal cell complexes, formed in vivo, have been isolated by others and postulated to play a role in T-cell differentiation. These previous studies have been hampered, however, by a time-consuming isolation procedure from which only small numbers of these complexes are recovered. We have examined a model to study thymocyte-stromal cell complexes in vitro in which thymocytes are added to primary cultures of thymus stromal cells. In the present study, we found that thymocytes were histotypically selective in their attachment to thymus stromal cells. We also investigated the kinetics of thymocyte attachment to these thymus stromal cells. Cultures were examined at selected time intervals from 5 min through 3 days of incubation. Thymocyte attachment to stromal cells was a biphasic interaction, with maximum surface attachment at 15 min of cocultivation, followed by migration of thymocytes into the cultures. Morphological studies were confirmed by using 3H-leucine-labeled thymocytes and liquid scintigraphy. With increased time in culture, thymocytes became amoeboid and migrated between the layers of stromal cells where thymocyte mitotic figures were seen at 4 and 8 hr. In some cases it appeared that stromal cells, which often grew two to three cell layers deep, played an active role in enclosing thymocytes within the cultures. Large numbers of viable thymocytes were observed in the cultures at 24 hr. The number of thymocytes then decreased progressively on days 2 and 3, when relatively few were found within the layers of the culture.

Animals↗

PubMed2002

Influence of calcium on Manduca sexta plasmatocyte spreading and network formation.

Plasmatocytes are a class of insect hemocytes important in the cellular defense response. In some species, they are phagocytic, protecting the insect from smaller pathogens. In many insects, they work in concert with other hemocytes (particularly other plasmatocytes and granular cells) to form nodules and to encapsulate foreign material. To perform these functions, plasmatocytes attach to, spread on, and surround suitable targets. Because of their importance, because we had previously observed that prolonged incubation of hemocytes in solutions containing the divalent cation chelator ethylenediaminetetraacetic acid (EDTA) inhibited plasmatocyte spreading, and because of the importance of divalent cations in many immune-related functions, we investigated the effect of calcium and magnesium on spreading of plasmatocytes from fifth instar Manduca sexta larvae. On glass slides, plasmatocytes spread more quickly and elongated in Grace's medium containing 5 mM calcium, compared to calcium-free medium. In the presence of calcium, plasmatocyte adhesion, spreading, and network formation were not visibly different in magnesium-free and magnesium-containing Grace's medium. Using immunomicroscopy with a monoclonal antibody specific for plasmatocytes, we measured the length and width of plasmatocytes incubated with several different concentrations of calcium. Plasmatocyte length positively correlated with calcium concentration to 5 mM (maximum concentration tested and approximately the hemolymph concentration). Mean plasmatocyte width was less in 0 and 5 mM calcium than in 0.05 or 0.5 mM calcium. On plastic, hemocytes survived longer than on glass (they survived beyond 24 h) and, in 5 mM calcium, formed an extensive network readily visible by phase-contrast microscopy. This network was never as extensive in the absence of calcium. Network formation in the absence of magnesium, but presence of calcium, resembled network formation in standard Grace's medium.

Animals↗

PubMed2001

Shape diversity among chick retina Müller cells and their postnatal differentiation.

It is currently believed that in each vertebrate species Müller cells in the central retina constitutes a fairly homogeneous population from the morphologic point of view and that particularly the chick Müller cell attains full shape differentiation at prenatal stages. However, in this study of the chick retina, from day 1 to day 55 of life, we show that there is a large variety of Müller cell shapes and that many of them complete shape differentiation postnatally. We used a cell dissociation method that preserves the whole shape of the Müller cells. Unstained living and unstained fixed cells were studied by phase-contrast microscopy, and fixed cells immunostained for intermediate filaments of the cytoskeleton were studied by fluorescence microscopy. Our results show that (1) Müller cell shapes vary in the origination of the hair of vitread processes, in the shape of the ventricular (outer or apical) process, in the presence or absence of an accessory process, as well as in the number and shape of processes leaving from the ventricular process at the level of the outer nuclear and outer plexiform layers (ONL/OPL); (2) during the first month of life, many Müller cells differentiate the portion of the ventricular process that traverses the ONL, most Müller cells differentiate the ONL/OPL processes, and all Müller cells differentiate the thin short lateral processes leaving from the vitread hair processes at the level of the inner plexiform layer (IPL). The number of cells differing in the shape of the ventricular process and that of cells with and without accessory process were estimated. The spatial relationship between the outer portion of the ventricular process of the Müller cell and the photoreceptor cells was also studied. Our results show that the branching of the ventricular process and the refinement of Müller cell shape is achieved without apparent participation of growth cones. We give a schematic view of how the branching of the ventricular process might take place and propose the size increase of photoreceptor soma as a factor responsible for this branching.

Aging↗

PubMed1988

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding.

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.

Acridine Orange↗

PubMed2005

Fidelity of micropatterned cell cultures.

Methods that enable the culture of micropatterned cells may help advance our fundamental understanding of cell-cell and cell-surface interactions, while facilitating the development and implementation of cell-based biological assays. However, the long-term stability of the cell patterns can limit the time scales over which such methods can be informative. Here we used self-assembling monolayers (SAMs) to localize the adsorption of baby hamster kidney (BHK-21) cells as well as cells from a murine astrocytoma-derived cell line (delayed brain tumor) in linear arrays. We tested the effects of surface chemistries, fibronectin pre-treatments, array dimensions, and cell types on pattern fidelity. Changes in patterns were monitored by phase-contrast microscopy up to 96 h post-plating, followed by digital imaging, and these changes were quantified by measuring an "intrusion distance" or the average distance cells extend beyond the initial adhesive/non-adhesive boundary. Loss of pattern boundaries involved different mechanisms for different cells. Treatment of patterned surfaces with fibronectin prior to plating of cells tended to promote earlier loss of pattern fidelity, and the extent of pattern loss was further augmented for SAMs formed using hydrophobic monolayers. Finally, reduction of gap spacing between adjacent cell arrays promoted pattern loss.

Adsorption↗