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PubMedDate not supplied

Excitochemical-induced trophic bridging directs axonal growth of transplanted neurons to distal target.

Brain grafts directly placed in their target regions often lack proper connections and normal regulation. When brain grafts are placed in their ontogenetic normal area, the axonal outgrowth of grafts to distal target regions is a major obstacle. We previously demonstrated that directional axonal growth of grafts can be facilitated by laminin. In this study we demonstrated that excitochemicals induced trophic environment can be strategically used as a bridge to guide transplanted neurons to innervate a distal target of millimeter distance, and has a unique effect on fiber expansion in the target region. To distinguish dopamine (DA) growth from that of transplants, 6-hydroxydopamine was unilaterally injected into medial forebrain bundle to remove dopamine innervation to the striatum. Ibotenic acid (IB), kainic acid (KA), or phosphate buffer (vehicle) were microinjected through a glass-pipet to make a 7 mm long tract between the substantia nigra and the striatum in Sprague-Dawley rats. Gestational 14-15 days brainstem slurry was transplanted either in the same or a separate tract into nigra at the bottom of the bridge. All transplants survived with abundant serotonin (5-HT) and DA neurons. Traceable DA fibers formed distinct bundles from grafts along the length of IB/KA tracts. Upon arrival at their major target regions, fibers from bundles were capable of leaving the tracts, and reinnervating the DA-vacated striatum. The 5-HT fibers formed bundles similar to DA fibers but left the tract earlier upon arrival at their major target the globus pallidus and when arriving at the striatum and cortex. Fibers from grafts with vehicle tracts grew randomly and not into the tract. These results indicate that the trophic effects of chemical lesioning serves as a remarkably effective axonal guidance for more than one type of fetal neuron to innervate distal brain regions. This method has great potential in directing fibers in many transplant models.

Animals↗

PubMed1990

Therapeutic drug monitoring analysis systems for the physician office laboratory: a review of the literature.

Commercially available systems for therapeutic drug monitoring in the physician office laboratory (POL) are reviewed. The Abbott Vision, Kodak Ektachem, Syntex AccuLevel, Syva Emit QST, Ames Seralyzer ARIS, and Ames Clinimate ARIS have been found to be sensitive and accurate compared with more conventional laboratory assays, and are well-suited for the POL. The number of available drug assay is very limited with every system except the QST. The QST offers a large menu and would be appropriate for large practices. The Abbott TDx or other semiautomated system usually found in the clinical chemistry laboratory may be particularly useful and cost-effective in office practices with a large volume of specimens. AccuLevel or ARIS might be the best choices for clinics or pharmacies that generate few samples. The start-up time is the longest with the Vision and Ektachem systems (30 minutes). Most systems have the capacity to produce results within one to five minutes with the exceptions of the Vision (13 minutes for theophylline) and AccuLevel (20 minutes). The Vision is the most automated system and both AccuLevel and the Vision use whole blood, thereby bypassing the additional time required for centrifugation of the patient specimen. The Ektachem, Vision, and QST have an operator-independent pipetting step that offers a significant advantage when operated by semiskilled personnel. Although the AccuLevel eliminates the need for a large initial capital expenditure, the individual cost per test is higher. Leasing programs are available or certain systems. Increased government regulation will improve the quality control of therapeutic drug monitoring in the POL.

Clinical Laboratory Techniques↗

PubMed2000

Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format.

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Cloning, Molecular↗

PubMed2000

High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence.

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.

Antibodies, Monoclonal↗

PubMed2001

Evaluation of liquid handling conditions in microplates.

Liquid handling in higher density microplates (e.g., 1536-well microplates) for more efficient drug screening necessitates carefully selected and optimized parameters. The quality of a liquid handling procedure is dependent on the carryover rate of residual liquids during the pipetting process, the mixing behavior in the wells, foam and bubble formation, and evaporation. We compared and optimized these parameters in 96-, 384-, and 1536-well microplates, and herein we critically evaluate the performance of the CyBi-Well 96/384/1536 automated micropipetting device, which formed the basis of our evaluation studies.

Drug Contamination↗

PubMed2001

High throughput screening method for identification of new lipofection reagents.

Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.

Animals↗

PubMed2003

Ionworks HT: a new high-throughput electrophysiology measurement platform.

To address the throughput restrictions of classical patch clamp electrophysiology, Essen Instruments has developed a plate-based electrophysiology measurement platform. The instrument is an integrated platform that consists of computer-controlled fluid handling, recording electronics, and processing tools capable of voltage clamp whole-cell recordings from thousands of individual cells per day. To establish a recording, the system uses a planar, multiwell substrate (a PatchPlate). The system effectively positions 1 cell into a hole separating 2 fluid compartments in each well of the substrate. Voltage control and current recordings from the cell membrane are made subsequent to gaining access to the cell interior by applying a permeabilizing agent to the intracellular side. Based on the multiwell design of the PatchPlate, voltage clamp recordings of up to 384 individual cells can be made in minutes and are comparable to measurements made using traditional electrophysiology techniques. An integrated pipetting system allows for up to 2 additions of modulation agents. Typical throughput, measurement fidelity, stability, and comparative pharmacology of a recombinant voltage-dependent sodium channel (hNav1.3) and a voltage-gated potassium channel (hKv1.5) exogenously expressed in CHO cells are presented. The IonWorks HT device can be used in biophysical and pharmacological profiling of ion channels in an environment compatible with high-capacity screening.

Animals↗

PubMed2006

A cost-effective solution to reduce dead volume of a standard dispenser system by a factor of 5.

A key trend in high-throughput screening is assay miniaturization to control reagent costs and increase throughput. For this purpose, liquid-handling devices are used that transfer nano-to low-microliter volumes into all currently used microtiter well plates. One drawback of many available dispenser and pipetting systems are high dead volumes. Therefore, the authors were looking for an easy and simple solution to modify their standard liquid-handling device, PerkinElmer's FlexDrop Precision IV, allowing for a dead volume reduction to receive maximum benefit from miniaturized assay formats. Internal reservoirs were developed and constructed by Schering's Technical Development Laboratory (TDL), which are directly connected to the dispenser banks of FlexDrop without tubing. Using these newly built reservoirs, the dead volume was decreased by a factor of 5 in comparison to the manufacturer's reservoirs without compromising liquid-handling parameters such as accuracy and precision. The modified system displayed a high robustness and reliability under routine high-throughput screening conditions.

Buffers↗

PubMed2006

Assay development and screening of a serine/threonine kinase in an on-chip mode using caliper nanofluidics technology.

Kinases are key targets for drug discovery. In the field of screening in general and especially in the kinase area, because of considerations of efficiency and cost, radioactivity-based assays tend to be replaced by alternative, mostly fluorescence-based, assays. Today, the limiting factor is rarely the number of data points that can be obtained but rather the quality of the data, enzyme availability, and cost. In this article, the authors describe the development of an assay for a kinase screen based on the electrophoretic separation of fluorescent product and substrate using a Caliper-based nanofluidics environment in on-chip incubation mode. The authors present the results of screening a focused set of 32,000 compounds together with confirmation data obtained in a filtration assay. In addition, they have made a small-scale comparison between the on-chip and off-chip nanofluidics screening modes. In their hands, the screen in on-chip mode is characterized by high precision most likely due to the absence of liquid pipetting; an excellent confirmation rate (62%) in an independent assay format, namely, filtration; and good sensitivity. This study led to the identification of 4 novel chemical series of inhibitors.

Drug Evaluation, Preclinical↗

PubMed1999

A Fluorescence-Based High Throughput Screen for the Transporter Associated with Antigen Processing.

The transporter associated with antigen processing (TAP) is essential for antigen presentation by major histocompatibility complex (MHC) class I molecules. Traditional methods used to analyze peptide transport mediated by TAP require radioactive labeling of peptides and time-consuming manipulation of Concanavalin A-Sepharose. Drug discovery research requires rapid and reliable evaluation of large number of samples for bioactivity. To meet these requirements a nonradioactive, HTS assay for peptide transport activity of TAP has been developed. The radioactive label in the traditional assays has been replaced by a fluorescent label without compromising the transport efficiency of labeled peptide or the sensitivity of the assay. The use of multiscreen filtration plates has facilitated higher throughput and eliminated the centrifugation steps used in traditional TAP assays. The HTS assay shows similar kinetic characteristics as compared to the traditional assay. The HTS assay has been adapted on a Quadratrade mark 96-320 96-channel pipetting station (Tomtec, Hamden, CT) by optimizing time course, dose response of TAP to peptides and adenosine triphosphate (ATP), signal/noise ratio, reproducibility, and reagent stability. This HTS system has been utilized to screen a multiplexed compound library with a maximum of throughput 17,600 compounds per week.

Journal Article↗

PubMed1979

The preparation of cervical scrape material for automated cytology using gallocyanin chrome-alum stain.

A method is described for preparing cervical scrape specimens for automated analysis on the Cerviscan prescreening system. In order to reduce the cellular clumping found in cervical scrape material, cells are collected in suspension, syringed to disaggregate the cell clumps, and then pipetted onto a glass to give a monolayer of cells. The cells are then stained with gallocyanin chrome-alum to give the required quantitation of nucleic acid content, using a rapid staining procedure. Experimental results are given which show that specimens prepared by this method are more suitable for automated analysis than the conventional Papanicolaou stained preparation.

Cell Separation↗

PubMed1994

Interaction of Pseudomonas aeruginosa with human respiratory mucosa in vitro.

Pseudomonas aeruginosa commonly infects the airways of patients with cystic fibrosis and bronchiectasis. It produces several toxins that slow ciliary beat, stimulate mucus production and damage epithelium. It adheres to epithelial cells, damaged mucosa (in animal models), and mucus. However, little is known of the interaction of P. aeruginosa with intact human respiratory mucosa. We have studied the interactions of a nonmucoid clinical isolate of P. aeruginosa with adenoid tissue in a novel organ culture model with an air-mucosal interphase P. aeruginosa (5.9 +/- 0.9 x 10(6) colony-forming units (cfu)) was pipetted onto the organ culture surface, and incubated for 15 min, 1, 2, 4, 8, 12, 16, and 24 h, at 37 degrees C in 5% CO2 in a humidified atmosphere. Assessment has been made by transmission and scanning electron microscopy. Transmission electron microscopy (TEM) showed that uninfected organ cultures had normal ultrastructure. TEM of infected organ cultures at 8 h showed significant epithelial damage: 43.9 +/- 10% of cells extruding from the epithelial surface, 17.7 +/- 3% of cells with loss of cilia, 32.9 +/- 10.2% of cells with mitochondrial damage, and 11.6 +/- 3% of cells with cytoplasmic blebbing. P. aeruginosa only infrequently adhered to normal epithelium, but adhered to areas of epithelial damage and to basement membrane. Scanning electron microscopy (SEM) of organ cultures up to 2 h found P. aeruginosa only infrequently associated with mucus. SEM at 4 h revealed P. aeruginosa predominantly associated with mucus and extruded damaged epithelial cells, but also occasionally associated with cilia, and very occasionally with unciliated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoids↗

PubMed2003

An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography.

BACKGROUND: Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. RESULTS: We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag) or glutathione S-transferase (GST)-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. CONCLUSION: A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies.His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.

Chromatography, Affinity↗

PubMed2006

High-throughput isolation of ultra-pure plasmid DNA by a robotic system.

BACKGROUND: With the availability of complete genomes, a systematic inventory of cellular processes becomes achievable. This requires assessing the function of all individual genes. Transfection of plasmid DNA into cell culture cells is an essential technique for this aim as it allows functional overexpression or downregulation of genes. While many robotic systems isolate plasmids for sequencing purposes, for more demanding applications such as transfections there is a shortage of robots for the high-throughput isolation of plasmid DNA. RESULTS: Here we describe a custom-made, automated device, which uses a special protocol to isolate plasmid DNAs with a purity sufficient for efficient transfections into mammalian cells. Approximately 1,600 ultra pure plasmids can be isolated in a 96-well plate format within 12 hours. As a unique feature the robot comprises the integration of a centrifuge instead of expensive columns, the use of a custom-made pipetting head with a movable gripper, especially designed shaking platforms and an acetone wash facility. CONCLUSION: Using this robot we demonstrate how centrifugation steps with multiple precipitations, most notably through a precipitation step of SDS in isopropanol, lead to high purity plasmid DNA and make possible high-throughput transfections into mammalian cells for functional gene annotations.

Chemical Fractionation↗

PubMed1980

Isolation of viable rat ventral prostate epithelial and nonepithelial cells.

A procedure is described for the dispersion, partial purification, and identification of epithelial and nonepithelial cells from the rat ventral prostate, the latter based in part upon the ability of cells to bind androgen. Since initial efforts to mechanically disrupt prostatic tissue at 2 C lysed many cells, minced rat prostates were exposed to collagenase at 37 C and further dispersed by repetitive pipetting and passage through a tissue sieve. Washed cells in culture medium were centrifuged through an isokinetic Ficoll gradient from which three visible fraction (1, 2, and 4) and a less definite fraction 3 were harvested. Characterically, fraction 1 contained cellular debris; fraction 2 contained round nucleated cells, fewer elliptically shaped cells, red blood cells, and rare free cell nuclei; fraction 3 contained somewhat larger elliptical and round cells; and fraction 4 contained larger round and elliptically shaped cells. These were epithelial cells, as judged by electron microscopy. Isolated prostate cells from rats castrated for 24 h were incubated with [3H]testosterone; 80-90% of the retained radioactivity, the majority of which was dihydrotestosterone, was associated with washed cells from fraction 4. Similar results were obtained after in vivo administration of labeled androgen and subsequent analysis of radioactivity in cells from these fractions. Histochemically and enzymatically demonstrable formalin-insensitive acid phosphatase was increased in bands 3 and 4, which were enriched in epithelial cells. Many cells in fractions 2-4 were viable before and after exposure to Ficoll, as estimated by their ability to exclude trypan blue, incorporate radioactive uridine into RNA, generate cell monolayers during 1-4 weeks of culture, and actively metabolize androgens. Compared to fraction 4, cells from fraction 2, considered to be enriched in nonepithelial cells, actively metabolized but bound much less [3H]testosterone and its metabolites. A number of epithelial cells in fraction 4 isolated from prostates dissociated at 2 C were associated with typical-C-type RNA viruses.

Animals↗

PubMed1976

Preparation of viable isolated bovine parathyroid cells.

A method is described for the preparation of isolated bovine parathyroid cells. Fresh, minced bovine parathyroid tissue is incubated with 2 mg/ml collagenase and 50 mug/ml DNAse for 1 h at 37 C with periodic pipetting. Parthyroid cells are separated from contaminating fat cells and red cells by low speed centrifugation. The resulting cell preparation is indistinguishable from parathyroid cells in intact bovine glands by light and electron microscopy. Hormone release is linear for at least 3 h at both high (2.0 mM) and low (0.5 mM) calcium concentrations and is inversely proportional to divalent cation concentration between 0.3 mM and 1.0 mM calcium as been observed previously both in vivo and in vitro. As in previous studies, hormone release is also stimulated up to 2-fold by 10(6) M (-)isoproterenol, an effect blocked by 10(-6)M (-)propranolol, suggesting a beta-adrenergic effect. Such a cell preparation should be useful for studying hormone binding and effects on cyclic nudleotides and cellular transport phenomena in both normal amd abnormal tissue.

Animals↗

PubMed2000

Gonadotropins and cytokines affect luteal function through control of apoptosis in human luteinized granulosa cells.

The luteal phase in the normal human menstrual cycle is known to be about 14 days. The physiological mechanisms that regulate the corpus luteum remain to be clarified, although apoptosis is reported to be involved. This study was undertaken to investigate the regulation of luteal function by gonadotropins, cytokines, and PGs, concentrating attention on the incidence of apoptosis and its molecular mechanisms in cultured human luteinized granulosa cells collected at oocyte pick-up from patients undergoing in vitro fertilization and embryo transfer. Clusters of granulosa cells were pipetted in 0.1% hyaluronidase in phosphate-buffered saline. After cell separation by centrifugation using Ficoll-Paque, 1 x 104 viable cells/mL in RPMI 1640 medium with 10% FCS were used for experimentation. Substances added were FSH (100 ng/mL), hCG (100 ng/mL), LH (100 ng/mL), interleukin-1beta (IL-1beta; 10 ng/mL), transforming growth factor-beta1 (TGFbeta1; 10 ng/mL), macrophage colony-stimulating factor (MCSF; 10 ng/mL), tumor necrosis factor-alpha (TNFalpha; 10 ng/mL), and PGF2alpha (10 ng/mL). After 24-h culture at 37 C under 5% CO2 and air, cells were fixed with 4% neutral buffered formalin and stained with Hoechst 33258. Apoptotic bodies were counted under a fluorescence microscope, and immunostaining was performed using anti-Fas, Fas ligand, Bcl-2, Bax, and p53 antibodies. Incidences of apoptotic bodies in the group without substance addition were 0.7 +/- 0.2% (0 h), 5.9 +/-0.6% (24 h), and 7.9 +/- 1.2% (48 h); spontaneous increase was significant at the latter time points. Defining the incidence at 24 h as 100%, values after treatment were: FSH, 57%; LH, 84%; hCG, 44%; IL-1beta, 76%; TGFbeta1, 52%; M-CSF, 50%; TNFalpha, 177%; and PGF2alpha, 147%. Significant suppression was observed with FSH, hCG, TGFbeta1, and M-CSF (P < 0.01). On the other hand, significant induction occurred with TNFalpha and PGF2alpha (P < 0.01). On immunostaining, the incidence of stained cells with anti-Fas, Fas ligand, Bax, and p53 antibody was increased after 24-h incubation without addition. This was reduced by hCG, TGFbeta1, and M-CSF. No stained cells were observed with anti-Bcl-2 antibody before or after incubation. In conclusion, our results suggest that both gonadotropins (FSH and hCG) and cytokines (TGFbeta1 and M-CSF) may be involved in the support of luteal function via suppression of apoptosis, and that TNFalpha and PGF2alpha may contribute to ovarian dysfunction and/or luteal regression via its induction in human luteinized granulosa cells. Our results also suggest that Fas, Fas ligand, p53, and Bax may play roles in this apoptosis controlled by hCG, TGFbeta1, and M-CSF.

Adult↗

PubMed1991

Evidence for an inequality in the forces that generate principal and reverse bends in sperm flagella.

The response of the mechanism initiating flagellar bends to imposed mechanical transients has been studied by holding the head of a sea urchin sperm in the tip of a sinusoidally vibrating micropipet and then displacing the micropipet laterally at a speed of up to 1.15 micron ms-1 for 1.5 beat cycle, without vibration, before resuming sinusoidal vibration with the initial phase, frequency and amplitude at the new location of the pipet. This transient displacement of the micropipet delays the initiation of the bend that was due to initiate 0.5 beat cycle after onset of the displacement. The amount of this delay increases with the speed of the displacement, for speeds up to 1 micron ms-1. Analysis of the flagellar waveforms during the transient showed that with imposed displacements at speeds of equal magnitude, the initiation of a principal bend was delayed to a longer extent than that of a reverse bend. At a micropipet speed of 0.75 micron ms-1, there was an average delay of 0.21 beat cycle in the initiation time of a principal bend as compared to a delay of only about 0.04 beat cycle in the initiation time of a reverse bend during displacements in the opposite direction. For both principal and reverse bends, the second bend due to initiate during the transient displacement initiated in most of the cases with no delay, regardless of the micropipet speed. Our results suggest that the force generated by microtubule sliding to initiated a new reverse bend is greater than that generated to initiate a principal bend.

Animals↗