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 757 records · Page 42Linked to original sources
PubMed1992

Filter paper equilibration as a novel technique for in vitro studies of the composition of airway surface fluid.

We describe techniques which we developed to study the composition and regulation of airway surface fluid (ASF). ASF from isolated equine tracheal mucosa was absorbed onto or equilibrated with small strips of ashless filter paper after timed incubations in a water-saturated, 37 degrees C environment. After expression of the fluid from the paper, constant-volume aliquots of the samples were pipetted with a microvolumetric pipette on a filmed grid, together with standards containing known concentrations for Na, Cl, K, Ca, S, and P. Analysis of the microdroplets by dispersive X-ray microanalysis on a scanning electron microscope demonstrated that ASF from "unstimulated" tracheal tissue is significantly altered compared with the Ringer solution which was used to rinse the mucosal surface before incubation, in that Na is significantly decreased (approximately 130 mM), whereas K is significantly increased (approximately 16 mM). Furthermore, when a beta-adrenergic agonist (isoproterenol 10(-4) M) was added to the serosal surface, the Na concentration significantly increased to values which approached Ringer solution (146 +/- 5.3 mM) and were significantly different compared with the Na values obtained in unstimulated tissues. In contrast, Na remained unchanged after addition of a cholinergic agonist (metacholine, 10(-4) M) to the serosal side of the tissue; however, as with beta-adrenergic stimulation, most other minor elements and especially K significantly decreased compared with their values in unstimulated tissue. These results clearly suggest that ASF composition is clearly controlled by active transport of the airway epithelium and may be influenced by neurohumoral stimulation.

Animals↗
PubMed1986

Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina.

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Delivery of high-quality biomarker assays.

Biomarker measurements now support key decisions throughout the drug development process, from lead optimization to regulatory approvals. They are essential for documenting exposure-response relationships, specificity and potency toward the molecular target, untoward effects, and therapeutic applications. In a broader sense, biomarkers constitute the basis of clinical pathology and laboratory medicine. The utility of biomarkers is limited by their specificity and sensitivity toward the drug or disease process and by their overall variability. Understanding and controlling sources of variability is not only imperative for delivering high-quality assay results, but ultimately for controlling the size and expense of research studies. Variability in biomarker measurements is affected by: biological and environmental factors (e.g., gender, age, posture, diet and biorhythms), sample collection factors (e.g., preservatives, transport and storage conditions, and collection technique), and analytical factors (e.g., purity of reference material, pipetting precision, and antibody specificity). The quality standards for biomarker assays used in support of nonclinical safety studies fall under GLP (FDA) regulations, whereas, those assays used to support human diagnostics and healthcare are established by CLIA (CMS) regulations and accrediting organizations such as the College of American Pathologists. While most research applications of biomarkers are not regulated, biomarker laboratories in all settings are adopting similar laboratory practices in order to deliver high-quality data. Because of the escalation in demand for biomarker measurements, the highly-parallel (multi-plexed) assay platforms that have fueled the rise of genomics will likely evolve into the analytical engines that drive the biomarker laboratories of tomorrow.

Biomarkers↗
PubMedDate not supplied

Reduced stability of prostate-specific antigen after long-term storage of serum at -20 degrees C.

The measurement of serum prostate-specific antigen (PSA) is widely used for the detection and management of patients with prostate cancer. Many studies on the validity of PSA as a marker for prostate cancer are performed on clinical samples that have been stored frozen for years. We have studied the stability of free (F), total (T) and complexed (C) PSA immunoreactivity and the proportion of free to total PSA (F/T) in serum after melting sera stored at -20 degrees C for 2 years and 2 weeks, respectively. In contrast to the decrease in PSA-F and F/T observed in fresh samples, PSA-C decreased and PSA-F increased in a time-dependent fashion after thawing samples that had been kept frozen for 2 years. This caused a net decrease in PSA-T and an increase in F/T. These results suggest that even though serum PSA is fairly stable during short-term storage, long-term storage at -20 degrees C reduces the stability of PSA immunoreactivity. Thus, results obtained on samples stored for prolonged times at -20 degrees C should be interpreted with caution. Because of the changes in PSA-F and F/T in both fresh and archival samples stored unfrozen, it is recommended that sera are melted only for the period required for pipetting the samples.

Biomarkers, Tumor↗
PubMedDate not supplied

Automated immunofluorometric assay for MUC1.

The aim of the present study was to establish a robust, reliable and fully automated immunofluorometric assay for the breast cancer serum marker MUC1. This would further serve as a prototype assay for evaluation of other MUC1 assays based on new antibody combinations. Using time-resolved fluorescence as tracer signal we developed an automated immunofluorometric assay for MUC1 (MUC1 IFMA). This assay was compared with two commercial assays. The CA15-3 EIA (CanAg) which use the same antibodies as the MUC1 IFMA, and the ETI-CA-15-3 K (Sorin) which use the original antibodies defining the CA 15-3 assay. The three assays showed comparable results. The coefficient of variation was below 10% from 9 to 2,400 kU/l for the MUC1 IFMA, from 15 to 250 kU/l for the CA15-3 EIA, and from 25 to 200 kU/l for the ETI-CA-15-3 K assay. At a specificity of 0.94 the overall diagnostic sensitivities for the MUC1-IFMA, CA15-3 EIA and ETI-CA-15-3 K assays were 0.40, 0.37, and 0.38, respectively. When applied to metastatic breast cancer, all assays had sensitivities close to 0.80. There was a close correlation (Spearman rank = 0.99) between results from the new assay and the CA15-3EIA. The new automated assay was not strictly immunometric as we could not achieve conditions where solid phase or tracer antibodies were in apparent excess. However, the assay performed well at a wide range of assay conditions. The automation, which minimizes imprecision in pipetting and handling of samples, and the high capacity of the AutoDELFIA instrument enabling measurement of all samples in a single run, were important aspects for establishing a reliable assay. The principle of the new automated immunofluorometric assay will be used as a rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay. This new automated immunofluorometric assay will be useful in the rapid and reliable evaluation of a wide range of monoclonal antibody combinations in our search for the optimal MUC1 assay.

Adult↗
PubMed1999

Adhesion to fibronectin regulates expression of intercellular adhesion molecule-1 on eosinophilic cells.

BACKGROUND: Adhesion molecules may play an important role in eosinophilic activation as well as adherence of extracellular matrix (ECM) including fibronectin (FN) in the inflamed focus. Tissue eosinophils expressed inter-cellular adhesion molecule-1 (ICAM-1, CD54), whereas peripheral blood eosinophils did not. The molecular mechanisms of ICAM-1 expression on eosinophils are not clear. We immunologically examined the effect of adherence to FN on the expression of adhesion molecules in an eosinophilic cell line (EoL-1). METHODS: EoL-1 cells, suspended at a concentration of 2 x 10(6) cells/ml, were cultured at 37 degrees C in BSA or FN-coated 24-well plates. After 4 h, the cells were removed by pipetting, and the expression of adhesion molecules was evaluated by immunofluorescence. RESULTS: Adherence of EoL-1 to FN enhanced ICAM-1 (CD54) expression on EoL-1 (FN vs. BSA: 84.76 +/- 17.90 vs. 43.73 +/- 7.60, mean fluorescent intensity). Regarding other adhesion molecules on EoL-1 (CD11a, CD18, CD49d), the expression was not significantly augmented by adherence to FN. CONCLUSIONS: We concluded that the signal via adhesion of FN regulates the expression of ICAM-1 on eosinophilic cells. This study suggests that eosinophilic adhesion to ECM via adhesion molecules plays an important role in the pathogenesis of allergic inflammation through eosinophilic functional changes, including regulation of expressive adhesion molecules such as ICAM-1.

Cell Adhesion↗
PubMed1992

Effect of H1-receptor blockade on anthralin inflammation.

The effect of terfenadine, an H1-receptor antagonist, on anthralin inflammation was studied in 12 subjects. Subjects were randomised to receive terfenadine 60 mg or placebo b.d. in a double-blind, cross-over study. Anthralin 5, 10 and 20 micrograms in 10 microliters chloroform was pipetted onto flexor forearm skin. Anthralin inflammatory oedema and erythema were measured using Harpenden calipers and a reflectance photometer, 48 h after anthralin application. After a 3-day washout period, placebo and terfenadine were restarted and the procedures repeated on the opposite arm. Anthralin erythema and oedema increased with dose but the dose-response curves showed no significant difference with or without terfenadine (p greater than 0.05). This study shows that H1-receptor blockade does not inhibit anthralin inflammation.

Adult↗
PubMed1988

Effect of anti plasminogen monoclonal antibodies on whole blood clot lysis.

Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPW1PG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPW1PG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length), 125I-fibrinogen was added to citrated whole blood (0.36% sodium citrate) to about 5,000 cpm/10 microliter sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23 degrees, and rotated at 16 rpm. 10 microliter samples were taken for radioactive counting before clotting by addition of 10 microliter 3.02M CaCl2 (100%-value), 45 min after clotting (0%-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1993

Development of a rapid and sensitive chromogenic heparin assay for clinical use.

A new sensitive chromogenic heparin assay is developed, which is well suited for clinical use. For the assay two reaction mixtures are required which can be lyophilized and reconstituted on the day of use. These reagents are stable during at least 6 h. Only two time-dependent pipetting steps are necessary. Any compound that inactivates thrombin, or can potentiate thrombin inactivation by an inhibitor, can be measured with this assay, including standard heparin, low molecular weight heparins, hirudin, alpha-NAPAP, pentosan polysulphate and dermatan sulphate. It is shown that heparin can be measured accurately in whole blood and in plasma. By addition of dextran sulphate to one of the reagents a platelet factor 4-insensitive assay is developed, so heparin can be measured even in blood that is partially activated and thus contains platelet factor 4 which neutralizes heparin.

Anticoagulants↗
PubMed1980

Automaticity and entrance block induced by focal depolarization of mammalian ventricular tissues.

Isolated canine interventricular septa were studied with standard microelectrode techniques. Focal automaticity was induced by applying depolarizing current through an extracellular pipet in contact with the right bundle branch (RBB) of the ventricular specialized conducting system. Automaticity appeared with depolarization to transmembrane potentials of -50 mV or less. The spontaneous activity was neither depressed nor accelerated when overdrive suppression was attempted. Activity originating within the focus propagated into fully polarized surrounding tissue. However, entrance block, phasically related to the spontaneous cycle length, was an intrinsic property of these foci. Early premature beats initiated outside the focus failed to enter the focus, but the resulting electrotonus delayed the next automatic beat. Late premature beats captured and thereby accelerated the focus. Thus, the automatic foci could be extrafocal activity. Consequently, continous pacing at various rates precipitated complex rhythms with fixed coupling. Similar foci with exit conduction, entrance block, and electrotonic modulation also were demonstrated in focally depolarized papillary muscles in feline septal preparations. The unique properties of focally depolarized areas in which spontaneous activity is generated at low membrane potentials provide a mechanism capable of generating a wide array of arrhythmias.

Animals↗
PubMed1997

Effect of salmeterol on Pseudomonas aeruginosa infection of respiratory mucosa.

We have studied the effect of salmeterol on both P. aeruginosa interactions with the mucosa of nasal turbinate organ cultures and on pyocyanin-induced (20 microg/ml) and elastase-induced (100 microg/ml) damage to nasal epithelial cells. Organ cultures were exposed to salmeterol either by preincubation with 4 x 10(-7) M salmeterol for 30 min or by pipetting 20 microl of 4 x 10(-7) M salmeterol onto the organ culture surface immediately prior to bacterial inoculation. Infected organ cultures (8 h) had significantly (p < or = 0.01) increased epithelial damage, and P. aeruginosa was predominantly associated with damaged epithelium and mucus. Salmeterol significantly (p < or = 0.02) reduced epithelial damage caused by infection and the total number of adherent bacteria (p < or = 0.05), but bacterial distribution on the mucosa was unchanged. Nasal epithelial cells incubated with pyocyanin (20 microg/ml) or elastase (100 microg/ml) for 3 h had significantly (p < or = 0.05) increased cytoplasmic blebbing and mitochondrial damage versus control values. Elastase also significantly (p < or = 0.05) increased cell projection and reduced the level of ciliation. Cells preincubated with salmeterol (2 x 10(-7) M) showed a significant reduction in some features of cell damage caused by both toxins, which was inhibited by the beta2-adrenoceptor antagonist propranolol. Our results indicate that salmeterol reduces P. aeruginosa-induced damage to both organ culture and nasal epithelium.

Adrenergic beta-Agonists↗
PubMed1991

Enrichment of subpopulations of respiratory epithelial cells using flow cytometry.

The application of flow cytometry to enrich airway epithelial cell subpopulations is described. A complementary epithelial cell preparative technique is also outlined. The ability of the airway epithelium to protect the lung from environmental insults results from a complex interaction among the different cells that form its matrix. The separation of the different epithelial cell types is an essential step in the studies of mechanisms of the controlling factors of cell repair, cell differentiation, and neoplastic transformation. Epithelial cells of the New Zealand white rabbit trachea are prepared using enzymatic digestion and microdissection. Small sections of tracheal wall are dissected into pieces approximately 10 mm2. The mucosa is dissected and placed in 0.15% hyaluronidase for 40 min at 22 degrees C. Mucus is removed, and the mucosa is then placed in 0.1% pronase at 37 degrees C for 30 min. With careful dissection, the epithelium can be dissected from the mucosa in 10-mm2 sheets. Sheets of epithelial cells are placed in 6 ml of an enzymatic solution containing collagenase, 0.2% bovine serum albumin, 0.04% soya bean trypsin inhibitor, 0.06 ml of 1 M Hepes buffer for 3 h at 37 degrees C. The cells are gently pipetted during the 3-h period, yielding a suspension of viable cells. Subpopulations of these different cell types are enriched using an Orthocytofluorograph 50111. A krypton ion laser was used for excitation of cells at 488 nm. Forward-angle and 90 degrees scatter were gated on the histogram. The purification of the ciliated, basal, and secretory cells was 90%, 97%, and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effect of pentoxifylline on the flow of polymorphonuclear leukocytes through a model capillary.

Pentoxifylline is a methylxanthine derivative used to increase blood flow in peripheral atherosclerosis. Pentoxifylline is known to increase whole blood filtration rate, and recent evidence suggests that pentoxifylline increases the filtration rate of polymorphonuclear leukocytes (PMNs). The purpose of this study was to directly observe and quantitate the effect of pentoxifylline on the flow of individual PMNs into a model capillary. Short-term incubation of human PMNs with 10 mM pentoxifylline inhibited cell activation, as judged by a significant reduction in the number of neutrophils forming pseudopods. Furthermore, incubation of PMNs from 6 healthy men with 0.1, 1.0 and 10 mM pentoxifylline significantly decreased the time required for individual cells to be aspirated into a 4 microns pipet under constant pressure by 16 +/- 5%, 21 +/- 7%, and 41 +/- 8%, respectively (mean +/- SEM, p less than or equal to 0.05), compared with control. These experiments are the first direct demonstration of increased deformability in neutrophils treated with pentoxifylline. The results are consistent with the hypothesis that the beneficial effect of pentoxifylline on microvascular perfusion is partly due to an inhibition of PMN stiffness and activation.

Adult↗
PubMed1991

A multilayer membrane system for blood plasma isolation for use in primary health care.

A new membrane-based device is described for the quantitative separation of plasma from blood cells using neither centrifugation nor pipetting. The concentration of cholesterol in the isolated plasma was determined using a newly developed instrument, the Primetest. The results were in good agreement with those obtained in the hospital laboratory.

Blood Specimen Collection↗
PubMed1994

Early effects of cerebellopontine angle compression on rabbit distortion-product otoacoustic emissions: a model for monitoring cochlear function during acoustic neuroma surgery.

A rabbit model was developed to simulate the effects of ischemia that may occur during surgical removal of tumors involving the cerebellopontine angle or internal auditory canal. Specifically, the internal auditory artery was visualized through a posterior craniotomy and mechanically compressed for repetitive 1-minute intervals with a micromanipulator-controlled glass pipet terminating in a smooth bead. The 2f1-f2 distortion-product otoacoustic emissions were used to monitor the susceptibility of cochlear function to compressive effects. Distortion-product otoacoustic emissions were measured during discrete preblock, block, and postblock periods to determine the time course of distortion-product otoacoustic emission reduction and its return to baseline levels after rapid obstruction and resumption, respectively, of the cochlear vascular supply. Comparisons during these times indicated that preblock distortion-product otoacoustic emission levels were very stable, often varying by less than 1 dB. Additionally, distortion-product otoacoustic emissions were very sensitive to brief vascular occlusions in that, within approximately 25 seconds of blockage onset, emission levels at all frequencies decreased at rates of about -1.5 dB/second. On alleviation of the occlusion, distortion-product otoacoustic emissions rapidly and completely returned to preblock levels with a delay of about 4 seconds and recovery slopes of about 10.5 dB/second. A notable finding in some animals was that early and reproducible variations in distortion-product otoacoustic emission levels occurred within 5 to 8 seconds of internal auditory artery compression. When present, these transitory changes in distortion-product otoacoustic emission levels acted as early warning signs for vascular compromise of cochlear function.

Animals↗
PubMed1991

A comparison of five devices for the bedside monitoring of heparin therapy.

Five instruments were tested for their capacity to monitor heparin therapy on whole blood at the bedside. The instruments were 512 Coagulation Monitor (Ciba-Corning), Thrombotrack (Nycomed), Automated Coagulation Timer (Hemotec), Hemochron-ACT and Hemochron-APTT (International Technidyne Corporation). Fifty subjects with various levels of heparinisation were tested on each instrument and were also assayed for antithrombin III, fibrinogen, haematocrit, platelet count and plasma heparin level. The results were compared with a reference APTT performed on the Automated Coagulation Laboratory 300R (Instrumentation Laboratories). The Hemochron-ACT correlated least well. The Hemotec and Thrombotrack were unsuitable in a clinical setting because of pipetting requirements, although the Thrombotrack did correlate well with the reference parameters. The 512 Coagulation Monitor was the simplest to use, but its maximum response corresponded to the midpoint of the reference APTT therapeutic range. The Hemochron-APTT was simple to use, had an adequate response range and correlated well with reference parameters.

Blood Coagulation Tests↗
PubMedDate not supplied

Effect of hemorrhage on medial collateral ligament healing in a mouse model.

BACKGROUND: Medial collateral ligament injuries heal by a scar response. HYPOTHESIS: Increased hemorrhage at the site of medial collateral ligament injury improves healing. STUDY DESIGN: Controlled laboratory study. METHODS: Ninety-six mice were divided into two groups. Group 1 mice underwent knee medial collateral ligament transection with the opposite knee as a sham-operated control and group 2 animals additionally had 0.25 ml of tail cut blood pipetted to the medial collateral ligament transection site and sham-operated opposite knee. Ligament specimens were harvested at 3, 7, 21, and 28 days. RESULTS: Immunohistochemical analysis demonstrated peak macrophage counts at day 7 in all transected specimens. Macrophage counts were higher in group 2 than in group 1 at all time points, with a statistically significant increase of macrophages noted at day 7. In situ hybridization demonstrated increased collagen gene expression, with peaks at 7 and 28 days after transection. Group 2 animals showed increased gene expression at all time points as compared with group 1, with a statistically significant increase noted at 7 and 28 days. Biomechanical testing demonstrated progressive healing at each time point. At 28 days, the load to failure was 67% that of the sham-operated knee. CONCLUSIONS: This study suggests there is an increased healing response with bleeding at the ligament injury site. CLINICAL RELEVANCE: Identification of the factors involved with increased healing may allow manipulation of the healing response in the clinical setting.

Animals↗
PubMedDate not supplied

Clonogenic colony-forming ability of flow cytometrically isolated hepatic progenitor cells in the murine fetal liver.

Stem cells are defined as cells having multilineage differentiation potential and self-renewal capability. Hepatic stem cells have aroused considerable interest not only because of their developmental importance but also for their therapeutic potential. However, their presence in the liver has not yet been demonstrated. With the use of a fluorescence-activated cell sorter (FACS) and monoclonal antibodies, we attempted to ascertain whether hepatic stem cells are present in the murine fetal liver. For this purpose, we optimized a cell isolation technique for FACS sorting of fetal liver cells. When isolated CD45 TER119 cells (the non-blood cell fraction in the fetal liver) were tested for their clonogenic colony-forming ability, mechanical dissociation (pipetting) was the most suitable cell isolation technique for FACS sorting. We confirmed that these colonies contained not only cells expressing hepatocyte markers but also cells expressing cholangiocyte markers. To identify hepatic stem cells, studies must focus on CD45TER119- cells in the murine fetal liver.

Animals↗