Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PIPET”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed2006

Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry.

In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, automation, and data analysis procedures. The high molecular weight matrix meso-tetrakis(pentafluorophenyl)porphyrin eliminates chemical noise in the low-mass range, a "brushing" spotting technique in combination with prestructured target plates enables fast preparation of homogeneous matrix crystals, and addition of Li+ leads to intense cationized drug species. Complex biological samples were cleaned up using a 96-well solid-phase extraction plate, and the purified samples were automatically spotted by a pipetting robot. To obtain a suitable data analysis procedure for the quantitative analysis of drugs by MALDI-TOF mass spectrometry, various data processing parameters were evaluated on our two model drugs lopinavir and ritonavir. Finally, and most importantly, it is shown that the above-described procedure can be successfully applied to quantify clinically relevant concentrations of lopinavir, an HIV protease inhibitor, in extracts of small numbers of peripheral blood mononuclear cells (1 x 10(6)).

Adult↗
PubMed2006

Microfluidic serial dilution circuit.

In vitro evolution of RNA molecules requires a method for executing many consecutive serial dilutions. To solve this problem, a microfluidic circuit has been fabricated in a three-layer glass-PDMS-glass device. The 400-nL serial dilution circuit contains five integrated membrane valves: three two-way valves arranged in a loop to drive cyclic mixing of the diluent and carryover, and two bus valves to control fluidic access to the circuit through input and output channels. By varying the valve placement in the circuit, carryover fractions from 0.04 to 0.2 were obtained. Each dilution process, which is composed of a diluent flush cycle followed by a mixing cycle, is carried out with no pipeting, and a sample volume of 400 nL is sufficient for conducting an arbitrary number of serial dilutions. Mixing is precisely controlled by changing the cyclic pumping rate, with a minimum mixing time of 22 s. This microfluidic circuit is generally applicable for integrating automated serial dilution and sample preparation in almost any microfluidic architecture.

Dimethylpolysiloxanes↗
PubMed2006

Parallel chemical dosing of subcellular targets.

To characterize the role of spatially heterogeneous signaling in cellular function, methods are required for differentially exposing distinct regions of individual cells to externally applied reagents. Although a range of standard approaches exists for generating localized chemical gradients in culture, including puffer pipet spritzing and photolytic release of caged effectors, each is limited in key respects. Here, we report development of a cell-dosing strategy that addresses these limitations, providing the means to create steep gradients of any aqueous-miscible compound at essentially unlimited numbers of sites in parallel. In this approach, cells are cultured on a micrometer-thick polymer membrane that serves as a barrier between two stacked laminar-flow channels: one containing the cell culture and the other serving as a reagent flow cell. By focusing a pulsed laser beam onto one or more selected membrane positions, micrometer-diameter pores can be ablated upstream of desired cellular targets. Nascent pores thus serve as ports of entry into the culture environment for reagent streams capable of modifying subcellular features at positions potentially hundreds of micrometers from ablation sites. Importantly, individual reagent streams also can be rapidly eliminated by photo-cross-linking a protein plug over a selected pore. This versatile strategy for dynamically reshaping the chemical microenvironments in which cells reside should be useful in a variety of cell biology applications, ranging from neurotrophic modulation of neurite pathfinding to stimulation of cellular networks.

Cell Differentiation↗
PubMed1997

Detecting biomolecules in picoliter vials using aequorin bioluminescence.

The quantitative determination of proteins in picoliter-volume vials is described. The assay is based on the bioluminescence of the photoprotein aequorin along with photon-counting detection. Using this approach, avidin can be detected at femtomole levels by taking advantage of its inhibitory effect on the bioluminescence signal generated by biotinylated recombinant aequorin. The picoliter vials were fabricated on glass substrates using a laser ablation technique. Parameters that affect the reproducibility of the assay such as the fabrication and calibration of the pipets, the fabrication of the vials, and the composition of the assay solutions were studied.

Aequorin↗
PubMed1990

Giant liposomes: a model system in which to obtain patch-clamp recordings of ionic channels.

Cell-size, giant liposomes have been formed by submitting a mixture of asolectin lipid vesicles and native membranes from Torpedo, highly enriched in acetylcholine receptor (AcChR), to a partial dehydration/rehydration cycle [Criado, M., & Keller, B. U. (1987) FEBS Lett. 224, 172-176]. Giant liposomes can be prepared in bulk quantities, in the absence of potentially damaging detergents or organic solvents, and their formation is mediated by membrane fusion phenomena. In fact, fluorescence microscopy and freeze-fracture data indicate that protein and lipid components of the initial membranes and lipid vesicles are homogenously distributed in the resulting liposomes. Giant liposomes containing AcChR have been used as a model to evaluate whether this system can be used to monitor the activity of ionic channels by using high-resolution, patch-clamp techniques. Excised liposome patches in an "inside-out" configuration have been used in this work. We find that the most frequent pattern of electrical activity in response to the presence of acetylcholine in the patch pipet corresponds to a cation-specific channel exhibiting a dominant conductance level and a sublevel of approximately 78 and 25 pS, respectively. Such channel activity exhibits the pharmacological specificity, ion channel activation, ion selectivity, and desensitization properties expected from native Torpedo AcChR. Thus, it appears that the giant liposome technique offers a distinct advantage over other reconstitution procedures in that it provides a unique opportunity to undertake simultaneous biochemical, morphological, and electrophysiological studies of the incorporated ionic channel proteins.

Animals↗
PubMed1976

Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties.

Purine-nucleoside phosphorylase (EC 2.4.2.1; purine-nucleosideorthophosphate ribosyltransferase) was purified to apparent homogeneity from Chinese hamster liver and kidneys and from V79 tissue culture cells. The enzymes from both sources appear to have identical structural and catalytic properties. A simple rapid radioisotope assay capable of detecting 0.1 nmol of product for both directions of the purine-nucleoside phosphorylase reaction is described using Bio-Rad Cu2+ Chelex in Pasteur pipet columns. At 37 degrees C the purified enzyme converts 60 mumol of guanine to guanosine per min per mg of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates that the enzyme is composed of identical subunits of 30 000 molecular weight. The native enzyme behaves as a mixture of dimers of 68 000 molecular weight and trimers of 89 000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme had a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94 000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 muM both hypoxanthine and guanine, 35 muM form guanosine, 50 muM for inosine, and 200 muM for both ribose 1-phosphate and phosphate. During isoelectrofocusing, the enzyme forms a single major band at a pI of 5.25.

Animals↗
PubMed1996

Chloride channel properties of the uncoupling protein from brown adipose tissue mitochondria: a patch-clamp study.

The uncoupling protein (UCP) from brown adipose tissue mitochondria possesses H+ and Cl- transport activities [reviewed in Klingenberg, M. (1990) Trends Biochem. Sci. 15, 108-112]. Being a member of a mitochondrial carrier family, the transport of H+ and Cl- is carrier-like, i.e., much slower as compared to channels. Here we report that UCP reconstituted into giant liposomes displays stable chloride channel properties under patch-clamp conditions. The transport inhibitors (GTP, GDP, ATP, and ADP) also inhibit this channel in a reversible way, showing that the channel activity is associated with UCP. The slightly inward-rectifying chloride channel has a unit conductance of approximately 75 pS in symmetrical 100 mM KCl and closes at high positive potentials on the matrix side of UCP. Channel gatings switch from slow open-closure transitions to fast flickerings as the holding potential increases over +60 mV. Substitution experiments reveal a strong discrimination against cations [P(Cl-)/P(K+) approximately 17] and a permeability ratio order of Cl- > Br- > F- > SCN- > I- > NO3- > SO4(2-) > HPO4(2-) > gluconate. Nucleotide inhibition studies indicate that 70% UCP molecules had its matrix side oriented outside in the giant liposomes. Fatty acids, pH, divalent cations (Ca2+ and Mg2+), and mersalyl do not influence these Cl- currents. The Cl- channel can be blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) from the matrix side of UCP. The data are consistent with a dimer consisting of two monomeric 75-pS Cl- channels or with a monomeric 150-pS channel having a 50% subconductance state. The channel current increases with Cl- concentration showing a typical saturation curve with Km approximately 63 mM and gmax approximately 120 pS (100 mM KCl in the pipet). The Cl- conductance measured under these conditions is 6 orders of magnitude higher than the Cl- transport activity reported earlier, suggesting that the UCP has the potential of behaving as an anion channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMedDate not supplied

First steps in robot automation of sampling and sample management during cultivation of mammalian cells in pilot scale.

The robot automation of sampling and the subsequent treatment and storage of aliquots during mammalian cell cultivations was investigated. The complete setup, the development and testing of the sampling device, the robot arm, and the cell imaging system are described. The developed sampling device is directly coupled to a pilot bioreactor. It allows the computerized sterile filling of cell broth into 50 mL sample tubes. After each sampling the whole tubing system is steam sterilized. For further off-line treatment a robot takes the sample to the different devices. This robot is equipped with a camera and a force/torque sensor. A color-based object recognition guides the arm in a complex surrounding with different illumination situations, enabling the robot to load the sampling device with tubes and take the sample to further devices. For necessary pipetting and refilling we developed a computerized device. Cells are automatically stained and counted using an imaging system. The cell number and viability is automatically saved in a process control system together with the on-line parameters. During several cultivations in 20 and 100 L scale these main components of the automation strategy were successfully tested.

Animals↗
PubMedDate not supplied

Generation of equally sized particle plaques using solid-liquid suspensions.

A device is presented for the generation of equally sized plaques of sensitive particles in a 96-well format. The resulting particle plaques can be used for the measurement of adsorption isotherms and uptake kinetics in protein chromatography or for immobilization reactions. The particle plaques are formed from suspensions with a vacuum device that is designed as a reusable sandwich module. The particles are retained by a mesh while the solvent is removed by the vacuum. As most particles used for protein chromatography are sensitive to mechanical stress and dehydration, the vacuum device is gentle enough to allow the use of these particles, thus eliminating the uncertainty of slurry preparation and pipetting. Apparatus characteristics and preparation procedures are described precisely. The physical intactness of the particles after the preparation procedure is proved by microscopic analysis. Data on the uniformity of the obtained resin plaques with respect to the reproducibility of their adsorption performance is given. Finally, adsorption isothermal and kinetic data of BSA on an ordinary HIC system obtained by high-throughput measurements are shown as an application example.

Adsorption↗
PubMedDate not supplied

Automated RNA selection.

In vitro selection can be used to generate nucleic acid ligands (aptamers) to target molecules ranging in size and structure from cations to cells. However, the selection process is repetitive and time-consuming. We have automated a protocol for in vitro selection using an augmented Beckman Biomek 2000 pipetting robot. The automated selection procedure requires the integration of four devices and the optimization of four molecular biology methods, and is one of the most complex automated protocols attempted to date. Initial attempts at selection yielded robust replication parasites, but optimization of the automated selection procedure suppressed the emergence of these parasites and led to the selection of true nucleic acid ligands. Automated selection can now be used to generate nucleic acid aptamers in days rather than weeks or months.

Base Composition↗
PubMedDate not supplied

Optimizing photo-embossed gratings: a gradient library approach.

Methodologies for the rapid screening of coating systems were developed and applied to photopolymer lacquers for photoembossing applications. Continuous and discrete gradient libraries were prepared with a gradient in grating period along the short axis and along the long axis, a gradient in exposure energy, development temperature, film thickness, photoinitiator concentration, or monomer to polymer mass ratio. Discrete gradient libraries consisted of arrays of rectangular films made by pipetting a certain amount of sample onto a chemically patterned substrate consisting of hydrophilic patches surrounded by hydrophobic, fluorinated barriers. The shape and height of the photoembossed gratings were measured using an automated AFM. Optimum grating height was obtained for a 20-microm period at intermediate exposure energies, photoinitiator concentrations, or both. Height improves with development temperature (max 110 degrees C), monomer-to-polymer ratio (max 55 wt % monomer), and film thickness. Surface topography can also be optimized, depending on any specific application.

Calorimetry, Differential Scanning↗
PubMed2002

Liquid chromatographic determination of fluoroquinolones in egg albumen and egg yolk of laying hens using fluorometric detection.

A liquid chromatographic method was developed for the determination of ciprofloxacin, enrofloxacin, and sarafloxacin at 10-200 ppb in both egg yolk and egg albumen of laying hens. Egg yolk or albumen was acidified with 1 M phosphoric acid followed by deproteination with acetonitrile and centrifugation. The supernate was pipetted out, and the remaining protein pellet was extracted three times with acetonitrile. The supernates were combined and concentrated at 50 degrees C to <0.7 mL. The final volume was adjusted to 2 mL with 0.02 M potassium phosphate buffer, pH 2.5. Separation of the analytes was achieved using reversed-phase HPLC with fluorometric detection. The recoveries were >80% and coefficients of variation <20%. After validation, the method was applied for use in a national survey for fluoroquinolones in table eggs. Of the 276 eggs assayed, none was found positive for fluoroquinolones. The findings suggest that illegal use of fluoroquinolones in laying hens is not widespread.

Animals↗
PubMed2005

Automated, high-resolution micropipet aspiration reveals new insight into the physical properties of fluid membranes.

We describe recent advances in our experimental approach to examine the physical properties of biological and artificial membranes by automated micropipet aspiration. New instrumentation allows us to apply fast yet precise tension protocols to membranes while continuously recording the membrane deformation with high-speed videomicroscopy. Robust algorithms for subpixel geometric measurements track the displacements of membrane edges with resolution of a few nanometers and enable us to determine changes of the membrane area and enclosed volume of pipet-aspirated cells or vesicles with exceptional accuracy. Experimentation and data analysis are greatly facilitated by custom-written software whose basic design is described here as well. Example measurements demonstrate how this technique has significantly improved the amount and reliability of data obtained by various types of micropipet-aspiration experiments, allowing us to study interesting aspects of membrane behavior that have eluded earlier techniques.

Algorithms↗
PubMed2006

Ejection dynamics of a simple liquid from individual carbon nanotube nozzles.

Molecular dynamics simulations show that the flow of a high pressurized atomic liquid inside carbon nanotube "pipets" occurs in one-atom-thick well-defined laminae. Fluxes and velocities at ejection are a function of the inlet diameter and the type of outlet. In the conditions investigated here, the force of the ejected liquid is similar in value to that of biomotors, while the output per second is of the order of picoliters.

Nanotechnology↗
PubMedDate not supplied

Self-contained on-chip cell culture and pretreatment system.

In this study, we describe a simple on-chip cell culture and pretreatment system that requires no external machines. Conventional cell culture utilizes culture dishes or microtiter plates, where pipetting and centrifugation are indispensable for washing cells and changing media. However, our microdevice requires no external centrifugation or pump. Utilizing this microdevice, we attained dramatically shorter total analytical time with a high-throughput screening system for proteomic analysis (1 min per 12 samples; one eightieth of the conventional time). Protein expression of Jurkat cells during stress-shock induced apoptosis was readily analyzed using this system. We found that a seaweed extraction effectively induced apoptosis of Jurkat cells.

Apoptosis↗
PubMed2006

Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics.

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.

Bacterial Proteins↗
PubMed2000

Evaluation of endothelial cell migration with a novel in vitro assay system.

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349-354). An aliquot of 15-20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor alpha (TNFalpha). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm(2) and 5.3 mm(2) for TNFalpha-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater - 6.4 mm(2) and 6.5 mm(2) with and without TNFalpha treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30-40% on vWF/collagen, and 60-80% on FN/collagen, with increased proliferation observed with TNF-alpha treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.

Cell Movement↗
PubMed1999

Evaluation of the HB-Quick: a portable hemoglobinometer.

OBJECTIVE: The Hb-Quick is a new portable hemoglobinometer that uses disposable cuvettes to measure the total hemoglobin concentration of capillary, venous, or arterial blood. Therefore, the objectives of this study were 1) to evaluate the performance of this compact, battery-powered hemoglobinometer by assessing its precision, accuracy, and linearity, 2) to determine whether its measurements suffer from interference by hemolysis, bilirubin, fetal hemoglobin, or hemodilution, and 3) to establish whether it can easily be used by clinical personnel with little or no laboratory training. METHODS: The precision of the test instrument was assessed by making repeated measurements on blood samples. Its accuracy and linearity were evaluated by comparing its measurements with the internationally accepted cyanmethemoglobin method. Samples of whole blood with and without bilirubin, fetal hemoglobin, hemolysis, and hemodilution were also analyzed to determine if any interference occurred when these disturbances were present, and it was placed in physicians' offices to evaluate its use by non-laboratory personnel. RESULTS: Repeated measurements on blood samples with a wide range of hemoglobin concentrations were consistent with the precision specification (0.25 g/dl). The bias of the new hemoglobinometer was calculated as the mean difference between its readings and measurements with the cyanmethemoglobin method, and its accuracy as the standard deviation of the differences between the two methods. As assessed in this manner, the new hemoglobinometer had a bias of -0.04 g/dl and an accuracy of 0.40 g/dl. The linearity was checked over a hemoglobin concentration range from 0 to nearly 30 g/dl. There was a highly significant linear relationship between its readings and measurements with the cyanmethemoglobin method (slope = 0.997, y-intercept = 0.005, r = 0.999). Complete hemolysis of the sample increased the readings on average by only 0.22 g/dl. Bilirubin (17.5 mg/dl) increased the reading by an average of 0.29 g/dl, and fetal hemoglobin (76.5% HbF) reduced the readings by an average of only 0.035 g/dl. Diluting blood samples with saline also did not appreciably affect its accuracy. CONCLUSIONS: The new hemoglobinometer is fast and easy to operate. No sample preparation or pipetting is required. To operate the instrument, the user simply allows a drop of blood to fill the disposable cuvette by capillary action and inserts the cuvette into the instrument. The instrument analyzes the 10 microl sample and displays the results in less than 10 seconds. The interference caused by hemolysis, hemodilution with saline, fetal hemoglobin, and bilirubin were too small to be of any dinical importance. Tests in physician's offices indicated that clinical personnel with little or no formal laboratory training could successfully use this device. The observed precision, accuracy, linearity, and freedom from interference indicate that this hemoglobinometer is suitable for near-patient testing in a wide range of clinical settings including physicians' offices.

Bilirubin↗