Multiwell Culture Plates Research Articles

Abstract 70: Mitochondrial localization of adiponectin receptor 1 (AdipoR1) induces apoptosis in cancer cells

Abstract INTRODUCTION Previous studies by us and others demonstrated tumor suppressive effects of adiponectin, an adipocyte derived hormone. Adiponectin signaling occurs through two distinct membrane receptors; AdipoR1 and AdipoR2. There is paucity of data how their activation leads to tumor suppression. Here we present data indicating localization of AdipoR1 to the mitochondria and its role in apoptosis of cancer cells. MATERIALS AND METHODS Human kidney (RCC1932) and cervical (HeLa CCL-2) cancer cells as well as human embryonic kidney cells (HEK 293) were used as models. Full length AdipoR1 and its N-terminal (ligand binding) and C-terminal (intracellular moiety) truncated mutants (mutAdipoR1) ORFs were subcloned into YFP expressing vectors (pEYFP- AdipoR1, pEYFPmutAdipoR1). Cells were seeded onto glass cover slips in multi-well culture plates and transfected with vectors through Lipofectamine-2000 (Invitrogen) method 24 h after seeding. The effect of adiponectin (BioVendor) supplementation was also tested. Immunocytochemistry staining were with MitoTracker labeling or anti-MnSOD (mitochondria), anti-Stearoyl-CoA desaturase (SCD-1, ER), anti-Reb5, anti-manose-6-phosphatase receptor (M6P, endosome); or anti-Catalase (peroxisome). Images of the cells were collected with DM IRB Deconvolution Microscope (Leica) or Confocal microscope LSM510 (Carl Zeiss). Apoptosis was determined by sub-diploid population detection and or annexin binding using flow cytometry. RESULTS Over-expression of AdipoR1 in the cells induced apoptosis which was further augmented by adiponectin treatment. This was evident only in cells expressing full length AdipoR1. Furthermore, cells expressing mutAdipoR1 had different intracellular distributions with loss of mitochondrial localization of the receptor and pro-apoptotic activity. CONCLUSIONS This is the first report showing that AdipoR1 can be localized in the mitochondria. Adiponectin induce apoptosis in cancer cells through mitochondrial localization related mechanism that requires both ligand and signaling domains of AdipoR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 70.

The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts

This study describes a medium-throughput system based on deposition of calcium phosphate films in multi-well tissue culture plates that can be used to study the effect of inorganic additives on the behavior of osteoblasts and osteoclasts in a standardized manner. All tested elements, copper, zinc, strontium, fluoride and carbonate were homogenously deposited into calcium phosphate films in varying concentrations by using a biomimetic approach. The additives affected morphology and composition of calcium phosphate films to different extent, depending on the concentration used. The effect on proliferation and differentiation of MC3T3-E1 osteoblasts depended on the compound and concentration tested. In general, copper and zinc ions showed an inhibitory effect on osteoblast proliferation, the effect of strontium was concentration dependent, whereas films containing fluoride and carbonate, respectively, augmented osteoblast proliferation. Copper and zinc had no effect or were mild inhibitory on osteoblast differentiation, while strontium, fluoride and carbonate ions demonstrated a clear decrease in differentiation in comparison to the control films without additives. Primary osteoclasts cultured on calcium phosphate films containing additives showed a significantly decreased resorptive activity as compared to the control, independent on the element incorporated. No cytotoxic effect of the elements in the concentrations tested was observed. The system presented in this study mimics bone mineral containing trace elements, making it useful for studying fundamental processes of bone formation and turnover. The present results can be used for modifying bone graft substitutes by addition of inorganic additives in order to affect their performance in bone repair and regeneration.

Practical histological methods for use with cultured cells

The histology laboratory can face many challenges when small, often critical, specimens of cultured cells are submitted for specialized immunocytochemical studies or special stains. Although clinical pathology labs often receive cell preparations, these usually contain enough cells so that pellets can be formed by centrifugation, and the pellets directly embedded and sectioned. Research labs, however, often need to submit very small samples of cells for experimental studies. We summarize here a number of techniques that currently are available and methods we have developed and/or adapted and used in our laboratory over the years. We describe the utility of multi-chambered slides for cell culture and histologic studies, multi-well cell culture plates, monolayer cell culture on specialized coated cell wells, cell well inserts, and agarose embedding techniques for small cultures of cells and for cultures that require antigen retrieval or multiple antibody localizations. Traditional double embedding techniques, such as the use of agar, are also cited.

Perfused multiwell plate for 3D liver tissue engineering

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.

Characterization of a novel phantom for three-dimensional in vitro cell experiments

A novel intensity-modulated radiation therapy (IMRT) phantom for use in three-dimensional in vitro cell experiments, based on a commercially available system (CIRS Inc., Norfolk, VA), was designed and fabricated. The water-equivalent plastic phantom can, with a set of water-equivalent plastic inserts, enclose 1–3 multi-well tissue culture plates. Dosimetry within the phantom was assessed using thermoluminescence dosimeters (TLDs) and film. The phantom was loaded with three tissue culture plates, and an array of TLDs or a set of three films was placed underneath each plate within the phantom, and then irradiated using an IMRT plan created for it. Measured doses from each dosimeter were compared to those acquired from the treatment planning system. The percent differences between TLD measurements and the corresponding points in the treatment plan ranged from 1.3% to 2.9%, differences which did not show statistical significance. Average point-by-point percent dose differences for each film plane ranged from 1.6% to 3.1%. The percentage dose difference for which 95% of the points in the film matched those corresponding to the calculated dose plane to within 3.0% ranged from 2.8% to 4.2%. The good agreement between predicted and measured dose shows that the phantom is a useful and efficient tool for three-dimensional in vitro cell experiments.

Bioactive Contaminants Leach from Disposable Laboratory Plasticware

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Stimulation effects of salts on growth in suspension culture of a mangrove plant, Sonneratia alba, compared with another mangrove, Bruguiera sexangula and non-mangrove tobacco BY-2 cells

Effects of a wide range of concentrations of four salts (NaCl, KCl, MgCl2, CaCl2) on cell proliferation were investigated in suspension culture of a mangrove plant, Sonneratia alba. The results were compared to those obtained with suspension culture of another mangrove species, Bruguiera sexangula and non-mangrove tobacco BY-2 cells. Effects of salts on suspension cells were determined with both large scale (LS) method using a 100 ml culture flask, and/or a small scale (SS) method using a 24-well culture plate. Settled cell volume (SCV), packed cell volume (PCV), wet weight (WW) and dry weight (DW) were measured in LS method and PCV was in SS method. High concentrations of NaCl (25–100 mM) and MgCl2 (25 mM) showed strong stimulatory effects on growth in S. alba suspension culture, and low concentrations of KCl, MgCl2 and CaCl2 (10 mM) also showed similar stimulatory effects. In contrast, a remarkable stimulatory effect was not observed in B. sexangula and tobacco BY-2 cells. Cationic ions are effective for the growth of suspension cells in both mangrove species S. alba and B. sexangula. The SS method using multi-well culture plate and micro-tube measurement method developed in this report, can be applied for analysis of factors for stress tolerance.

Good statistical practice in pharmacology Problem 2

This paper is intended to assist pharmacologists to make the most of statistical analysis and in avoid common errors. A scenario is presented where an experimenter performed an experiment to test the effects of two drugs on cultured cells. Analysis of the results, expressed as percentage of control, by a one-way ANOVA yielded P=0.058 and the experimenter concluded that neither drug was effective. The data were expressed as percentage of control because of pairing of the data within each experimental run, a common feature in cell culture experiments. Such data can be analysed with potentially more powerful ANOVA methods equivalent to the paired t-test. Monte Carlo simulations are presented to compare the power of relevant analyses. For data correlated within experimental run (i.e. paired values), transformation to percentage of control improved the power of a one-way ANOVA to detect a real effect, but a randomized block ANOVA (equivalent to a 2-way ANOVA with experiment and treatment as factors) using the raw values was substantially more powerful. The randomized block ANOVA performed well even with uncorrelated data, being only marginally less powerful than the one-way ANOVA. A randomized block ANOVA is far superior to the one-way ANOVA with correlated data, and with uncorrelated data it is only marginally less powerful. Thus where there is, or might reasonably be, such a correlation (e.g. relatedness among the data within a single experimental run, or within a multi-well culture plate, or within an animal, et cetera), use the more powerful randomized block ANOVA rather than one-way ANOVA.

Electrical stimulation enhances viability of human cutaneous fibroblasts on conductive biodegradable substrates

Electrically conductive biodegradable polymer membranes were prepared by mixing conductive polypyrrole particles with poly(L-lactic acid) solution followed by solution casting and solvent evaporation. Multi-well electrical cell culture plates were fabricated to host electrically stimulated cell culture and monitor parameters. Human cutaneous fibroblasts were cultured on conductive membranes with or without electrical stimulation (ES). Cell count, MTT, Hoechst staining, and SEM were performed to characterize the cells. The membranes supported the adhesion and proliferation of the fibroblasts in both the presence and absence of ES. In the presence of direct electrical field strength of 100 mV/mm, cell viability on the PPy/PLLA membranes at 2 and 24 h was 2.2- and 4.0-fold (p < 0.05) respectively of that on the same membranes without ES. Direct electrical current ranging from 2.5 to 250 microA/mm had no effect on the viability of cells cultured on the gold-coated Petri dish. Electrical field applied to conductive biodegradable polymer surfaces is therefore an effective approach to upregulate the mitochondrial activity of human skin fibroblasts.

Multiplexed expression and screening for recombinant protein production in mammalian cells.

BackgroundA variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.ResultsA nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.ConclusionThe combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

The Cell Adhesion Molecule L1 Is Required for Chain Migration of Neural Crest Cells in the Developing Mouse Gut

During development, the enteric nervous system is derived from neural crest cells that emigrate from the hindbrain, enter the foregut, and colonize the gut. Defects in neural crest migration can result in intestinal aganglionosis. Hirschsprung's disease (congenital aganglionosis) is a human condition in which enteric neurons are absent from the distal bowel. A number of clinical studies have implicated the cell adhesion molecule L1 in Hirschsprung's disease. We examined the role of L1 in the migration of neural crest cells through the developing mouse gut. A variety of in vitro and in vivo assays were used to examine: (1) the effect of L1 blocking antibodies or exogenous soluble L1 protein known to compromise L1 function on the rate of crest cell migration, (2) the effect of blocking L1 activity on the dynamic behavior of crest cells using time-lapse microscopy, and (3) whether the colonization of the gut by crest cells in L1-deficient mice differs from control mice. We show that L1 is expressed by neural crest cells as they colonize the gut. Perturbation studies show that disrupting L1 activity retards neural crest migration and increases the number of solitary neural crest cells. L1-deficient mice show a small but significant reduction in neural crest cell migration at early developmental stages, but the entire gastrointestinal tract is colonized. L1 is important for the migration of neural crest cells through the developing gut and is likely to be involved in the etiology of Hirschsprung's disease.

Establishment of primary cultures of rat olfactory bulb under serum-free conditions for studies of cellular injury

Olfactory dysfunction has been implicated in various neurodegenerative diseases including Parkinson's and Alzheimer's disease but, despite intense interest in the neurobiology of the olfactory bulb (OB), studies of neurodegenerative mechanisms have not been attempted in primary OB cultures. This study was aimed at developing a primary OB culture under serum-free conditions in order to investigate injury and excitotoxicity in vitro. Olfactory bulbs from rat pups were rapidly trypsinised and mechanically dissociated and the resultant single cell suspension was centrifuged through a high bovine serum albumin concentration gradient to reduce cellular debris before being seeded in multi-well culture plates. Cells were plated in neurobasal medium containing 0.5 mM glutamine, 25 mM K(+), 2% B27 and 10% fetal calf serum (FCS) for 24 h and, after 1 day in vitro (div1), were maintained without FCS. At div8, neurones exhibited extensive neuritic networks, were present as a monolayer and were mainly bipolar and immunopositive for gamma-aminobutyric acid indicating that they were intrinsic OB neurones. At div8, neurones (positive for microtubule-associated protein-2, 73%) predominated over astrocytes (positive for glial fibrillary acidic protein, 27%). Cellular injury produced by staurosporine, hydrogen peroxide and kainate, when assessed by morphological and biochemical procedures, was shown to be concentration-dependent and significantly reduced the numbers of neurones and astrocytes. Further analyses of kainate-induced injury revealed the presence of TUNEL-positive cells (indicative of apoptosis) and increases in intracellular free calcium, both of which were antagonised by CNQX. Thus, the serum-free culture developed here is amenable to morphological and high throughput neurochemical analyses of mechanisms contributing to the injury of OB neurones in vitro.

Caco-2 cell permeability assays to measure drug absorption

Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. When cultured as a monolayer, Caco-2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of compounds across the monolayer. In addition, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances. In many respects, the Caco-2 cell monolayer mimics the human intestinal epithelium. One of the functional differences between normal cells and Caco-2 cells is the lack of expression of the cytochrome P450 isozymes and in particular, CYP3A4, which is normally expressed at high levels in the intestine. However, Caco-2 cells may be induced to express higher levels of CYP3A4 by treatment with vitamin D3. Caco-2 cell monolayers are usually cultured on semipermeable plastic supports that may be fitted into the wells of multi-well culture plates. Test compounds are then added to either the apical or basolateral sides of the monolayer. After incubation for various lengths of time, aliquots of the buffer in opposite chambers are removed for the determination of the concentration of test compounds and the computation of the rates of permeability for each compound (called the apparent permeability coefficients). Although radiolabelled compounds were used in the original Caco-2 cells monolayer assays, radiolabelled compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS). Mass spectrometry not only eliminates the need for radiolabelled compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells.

Heart valve cryopreservation: Protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity

Aim To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity. Methods There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 °C. Dimethyl sulphoxide (Me 2SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks’ balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me 2SO in multi-well tissue culture plates at 1 °C/min to −80 °C. After storage overnight, the valve leaflets were warmed at approximately 11 °C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann’s solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth. Results After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me 2SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me 2SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks’ solution (Group 3) ( χ 2, p = 0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants ( χ 2, p = 0.007). The rates of cell growth in Group 2 (two-step addition of Me 2SO) and Group 3 (one-step addition of Me 2SO with additional Hanks’ solution rinse) were similar and faster than the Group 4 (one-step addition of Me 2SO without the additional Hanks’ rinse). Conclusion Single-step addition of Me 2SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks’ solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me 2SO, and the 5% Me 2SO step in the two-step protocol, merely served to reduce this carryover.

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.

A preliminary optical and electron microscopic study of the beta(1) integrin distribution pattern of human osteosarcoma-derived cells.

Immunogold labelling was used to study the organisation of the beta(1) integrins on osteosarcoma-derived osteoblasts (Saos-2 and MG-63). Monolayers of cells were prepared in multiwell culture plates on both uncovered and collagen-covered coverslips, and beta(1) integrins were primarily labelled using mouse monoclonal antibodies to beta(1) integrins. Indirect immunofluorescence labels using an anti-mouse fluorescein-conjugated goat antibody showed an even distribution of the beta(1) integrins on the cell membranes of all cell types used. A concentration of 2 microg/ml of the primary antibodies and a 1:100 dilution of the secondary antibodies were determined as the optimal concentration for labelling to use with indirect localisation of the primary antibodies gold conjugated to goat anti-mouse antibodies and viewed under an electron microscope. Ten nanometre gold particles were used for transmission electron microscopy (TEM) and 40 nm gold particles for scanning electron microscopy. TEM showed that beta(1) integrins were mainly clustered on the cell membrane processes with less labelling on the cell membranes themselves. The distribution of beta(1) integrins on osteosarcoma cells supports the concept that integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes.

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.

Development of novel elongated fiber-structure in protoplast cultures of Betula platyphylia and Larix leptolepis

Novel elongated fiber-structures were repeatedly found both in leaf protoplast culture of two clones of Betula platyphylla and in protoplast culture of embryogenic cells of Larix leptolepis. Suboptimum culture conditions for cell division appeared to lead to fiber formation when using multi-well plate culture with varying medium compositions The suboptimum conditions for cell divisions were brought about by (1) plant growth regulators: auxins and cytokinins; (2) pH: 3.5, 4.5, 5.8; (3) divalent cations: CaCl2 and MgCl2; and (4) sugars: sucrose and mannitol. Divalent cations had the most profound effect on fiber formation. Calcium ions were preferred by Betula and magnesium ions were preferred by Larix. Single fiberpurification and micro-staining methods using a micromanipulator were developed. The fibers fluoresced when stained with Calcofluor White and Aniline Blue, which suggested that they were composed of cell wall component(s), including callose (β-1,3-glucan). Electron microscopy showed that fiber bundles of Larix fibers had helical substructures.

Glucose-induced Metabolic Oscillations Parallel Those of Ca2+ and Insulin Release in Clonal Insulin-secreting Cells: A MULTIWELL APPROACH TO OSCILLATORY CELL BEHAVIOR

Insulin secretion from glucose-stimulated pancreatic beta-cells is oscillatory, and this is thought to result from oscillations in glucose metabolism. One of the primary metabolic stimulus-secretion coupling factors is the ATP/ADP ratio, which can oscillate as a result of oscillations in glycolysis. Using a novel multiwell culture plate system, we examined oscillations in insulin release and the ATP/ADP ratio in the clonal insulin-secreting cell lines HIT T-15 and INS-1. Insulin secretion from HIT cells grown in multiwell plates oscillated with a period of 4 min, similar to that seen previously in perifusion experiments. Oscillations in the ATP/ADP ratio in cells grown under the same conditions also occurred with a period of 4 min, as did oscillations in [Ca(2+)](i) monitored by fluorescence microscopy. In INS-1 cells oscillations in insulin secretion, the ATP/ADP ratio, and [Ca(2+)](i) were also seen, but with a shorter period of about 1.5 min. These observations of oscillations in the ATP/ADP ratio are consistent with their proposed role in driving the oscillations in [Ca(2+)](i) and insulin secretion. Furthermore, these data show that, at least in the clonal beta-cell lines, cell contact or even circulatory connection is not necessary for synchronous oscillations induced by a rise in glucose.

Induction of reduced endothelial permeability to horseradish peroxidase by factor(s) of human astrocytes and bladder carcinoma cells: detection in multi-well plate culture.

Endothelial permeability to horseradish peroxidase (HRP) was assayed in multi-well plate culture. Confluent cobblestone-monolayers of endothelial cells were incubated with HRP, and the amounts of HRP that permeated the monolayer and reached the bottom substrate were estimated by extraction of HRP with deoxycholate and following addition of chromogenic substrate of HRP into the extracts. Using this in situ assay method, we detected the reduced permeability to HRP in aortic and brain microvascular endothelial cells after 4-5 days culture with conditioned media of human astrocytes or bladder carcinoma cells. Preliminary characterization of the active factors(s) released from these cells was performed. This method will be useful for monitoring impermeable endothelial cell monolayers and identifying the active factor(s).