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 415 records · Page 23Linked to original sources
PubMed1987

Effect of rapid addition and dilution of dimethyl sulfoxide and 37 degrees C equilibration on viability of rabbit morulae thawed rapidly.

The aim of the present study was to examine the effects of various conditions of addition and dilution of dimethyl sulfoxide (Me2SO) and 37 degrees C equilibration, and also the effects of freezing in the solution which was prepared in advance and stored in plastic straws at -20 degrees C on the viability of rabbit morulae thawed rapidly. The embryos were cooled from room temperature to -30 degrees C at 1 degree C/min in the presence of 1.5 M Me2SO using a programmable liquid nitrogen vapor freezing machine with an automatic seeding device, then cooled rapidly, and stored in liquid nitrogen. The frozen straws were thawed rapidly (greater than 1000 degrees C/min). When Me2SO was added in a single step, equilibrated with embryos at 37 degrees C for 15 min and diluted out in a single step, a very high survival was obtained: transferable/recovered, 90%: developed/recovered, 96%. When embryos were pipetted into 1.5 M Me2SO that was prepared in advance, stocked in straws at -20 degrees C, and cooled, the proportions of transferable and developed embryos were equivalent to those of embryos frozen in the solution that was prepared immediately before use.

Animals↗
PubMed1985

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode.

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.

Acrosome↗
PubMed1991

Neuromuscular transmission to identified primary and secondary myotubes: a reevaluation of polyneuronal innervation patterns in rat embryos.

The early morphogenesis of rat skeletal muscle is a biphasic process involving two sequentially generated populations of myotubes. A small population of primary myotubes appears early and is followed by a much larger population of secondary myotubes which appear progressively over a number of days. All previously published electrophysiological studies of developing muscle have failed to appreciate the relevance of biphasic myotube production. Here we reevaluate the status of early motor innervation, taking into account the wide range of sizes and levels of maturity within the two myotube populations. Evoked end-plate potentials (EPPs) were recorded from fibers of E17-20 rat sternocostalis muscles. Impaled fibers were then marked by ejection of HRP from the recording pipet, enabling ultrastructural identification of fibers from which recordings had been made. The average number of synaptic inputs per fiber increased to a peak at E19, and the rate of rise of the EPPs increased with age. The majority of impaled fibers (76%) were subsequently found to be primary myotubes, even at ages when secondary myotubes formed the majority of fibers in the muscle. Electrophysiological studies during early stages of secondary myotube development therefore sample largely from the more mature primary fibers and probably give the wrong impression of the extent and degree of polyneuronal innervation and of synaptic rearrangement within the muscle. In addition, the results show that very young secondary myotubes are distinguished by EPPs of longer latency, slower rate of rise, and smaller size than those of other types of myotubes. These results suggest that young secondary myotubes are predominantly activated by EPPs which originate in adjoining primary myotubes and propagate electronically to the secondary myotube. We propose a new model of early synaptic rearrangement which accommodates the biphasic nature of muscle development. We also suggest that secondary myotubes do not require direct neural input for the initiation of their development.

Animals↗
PubMed1983

Effect of retinoic acid on cell-cell adhesiveness in cloned BHK21/C13 cells which form piling-up colonies.

The effect was studied of retinoic acid (RA) on cell-cell adhesiveness in Ag8-1 cells, which are piling-up colony-forming cells cloned from a Syrian hamster kidney fibroblastic cell line BHK21/C13. From the piled-up part of the colonies grown with RA (10 microM), many cells were dissociated by mere shaking or pipetting. The dissociated cells soon adhered to and spread on plastic surfaces in the presence of RA. The number of cells per colony increased almost at the same rate in the presence or absence of RA. The effect of RA on the appearance of cells dissociable from colonies was noticeable above 0.1 microM, prominent from 1 to 10 microM, greater when added in the earlier stages of colony formation and negligible when added just before the dissociation assay. Single cells from the monolayer culture grown with RA (10 microM) had less tendency to aggregate than did those from the control culture. Cells from the colonies grown with RA adhered to and spread on a plastic dish for bacterial use, but control cells seldom adhered. These results indicate that RA decreases the cell-cell adhesiveness or suppresses the development of it but increases cell-substratum adhesiveness.

Animals↗
PubMed1989

The role of transgenic animals in the analysis of various biological aspects of normal and pathologic states.

The introduction of foreign genes into the germ line of mammals has been a practical reality now for a number of years. This form of experimentation allows the creation of lines of animals tailor-made to answer specific molecular genetic questions. Manipulation of the mammalian embryos has been enormously important in developmental biology in recent years and that experience has brought about the possibility of new experiments allowing the molecular analysis of many biological processes. The methodologies involved in constructing transgenic animals have been published extensively in a number of comprehensive reviews. In typical experiments, pronuclear stage (one cell) embryos are collected after fertilization, but prior to the onset of cleavage. Exogenous cloned linearized DNA is injected into one of the two pronuclei by means of a finely drawn injection pipet. The manipulated embryo is transferred into the oviduct or ovarian bursal space of a surrogate mother previously mated with a sterile male. Alternative methods include retroviral transfection of cleavage stage embryos or insertion of genetically engineered embryo-derived embryonal stem cells into blastocysts. Offspring from these procedures are screened by standard molecular analyses to determine presence of the foreign genetic material. The present report explores the application of this methodology to a specific set of problems: (i) regulation of gene expression in vivo, (ii) the establishment of disease models for the study of pathogenesis, (iii) the use of exogenous genetic elements to correct specific genetic defects, (iv) the role of insertional mutagenesis in disruption of normal development, (v) analysis of genetic ablation, (iv) the use of transgenic animals to modulate carcinogens.

Animals↗
PubMed1992

Laminin-induced process outgrowth from isolated fetal rat C-cells.

A method for isolation of C-cells from rat fetuses was developed, and the morphological plasticity of the cells in primary culture systems was tested. Thyroid-parathyroid-ultimobranchial body (UB) complexes from 16-day rat fetuses were treated with 0.1% collagenase and 1000 PU/ml Dispase at 37 degrees C for 1 h. After dissociation by pipetting, UBs were obtained as remaining cell aggregates with diameters of 150-200 microns. The isolated UBs were cultured on untreated, fibronectin-coated, or laminin-coated substratum in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (1:1) supplemented with 5% fetal calf serum. In some experiments, the medium was changed to serum-free medium after 24 h of incubation, until the UBs had formed cell sheets. At Day 4 in vitro, the cultures were subjected to immunostaining using anti-calcitonin antiserum. On untreated or fibronectin-coated substratum, most of the C-cells exhibited polygonal or ovoid shapes, and 5-8% of them were found to project processes. On laminin-coated substratum, the ratio of process-bearing C-cells to total C-cells was 23% in serum-supplemented medium and 51% in serum-free medium. The longest processes reached 150 microns in length. The processes were intensely reactive with anti-alpha-tubulin antibody and were completely disintegrated by colcemid, suggesting that the microtubule cytoskeleton participated in the maintenance of the processes. Thus it was demonstrated that fetal rat C-cells are still responsive to environmental signals, such as laminin, and extend neuritic processes.

Animals↗
PubMed1983

Electrofusion of myeloma cells on the single cell level. Fusion under sterile conditions without proteolytic enzyme treatment.

A technique is presented which allows electrofusion of single cells under sterile conditions. The electrofusion chamber is placed in a Petri dish. Before a droplet of the fusion medium is pipetted between the electrodes, the chamber is completely covered with vaseline, which prevents the fusion medium evaporating. Additionally, the fusion chamber is treated with solutions containing poly(L)-lysine and pronase which results in a decreased movement of the cells on the glass between the electrodes and which allows electrofusion without any proteolytic pretreatment.

Animals↗
PubMed1988

Automated Sanger dideoxy sequencing reaction protocol.

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.

Autoanalysis↗
PubMed1978

A highly sensitive and reproducible microcytotoxicity assay for demonstrating cytotoxic antibodies to cell surface antigens.

An assay is described, in which target cells are suspended in diluted rabbit serum (as a source of complement) and pipetted into the wells of Falcon No. 3034 Microtest plates, to which diluted antiserum (or control serum) has been added. After 18--24 h of incubation, the plates are stained with crystal violet and counted using an electronic Quantimet image analyzer. This technique could reproducibly detect complement-dependent cytotoxic antibodies to target cell H-2 antigens and to MuLV gp70 on cell lines which expressed this antigen, at serum dilutions up to 1:160,000. The cytotoxic antibodies could be removed by absorption of sera with cells carrying the respective antigens.

Animals↗
PubMed1980

Detection of immunoglobulin-secreting lymphocytes by the use of a hemolytic plaque assay in liquid phase.

This paper describes a modification of the reverse hemolytic plaque assay allowing the enumeration of immunoglobulin (Ig)-secreting lymphocytes at rest or after in vitro stimulation. Ig-secreting cells are mixed with anti-Ig coated sheep red blood cells, developing anti-Ig antiserum and complement in liquid medium. This mixture is pipetted between two slides and the hemolytic plaques are numerated after incubation at 37 degrees C. The class of Ig secreted can be determined using a monospecific developing antiserum. This technique is easier to perform and more economical than the previously described method using agarose without loss of sensitivity, the number of plaques detected being about the same and the kinetic of their appearance comparable.

Hemolytic Plaque Technique↗
PubMed1981

Improvement of the protein A plaque assay for immunoglobulin secreting cells by using immunoglobulin-depleted guinea pig serum as a source of complement.

This paper describes a modification of the protein A hemolytic plaque assay for the enumeration of immunoglobulin (Ig)-secreting cells independent of antibody specificity of the Ig. This assay was originally developed by Gronowicz et al. (1976), and is based upon binding of the Fc portion of IgG to protein A. Ig-secreting cells are mixed with protein A-coated sheep erythrocytes, developing rabbit anti-Ig antiserum and guinea pig serum as a source of complement. This mixture is either pipetted between two microscope slides, or added to agarose and plated on a petri dish or microscope slide. The hemolytic plaques are enumerated after incubation at 37 degrees C. Here we show that purification of the guinea pig complement over a Sepharose-protein A column in order to eliminate the IgG fraction facilitates plaque formation. This modification reduces the incubation period required for plaque formation, and yields a higher number of, and more discrete plaques, than the original method.

Animals↗
PubMed1982

Enrichment (and depletion) of human suppressor cells with monoclonal antibodies and immunoglobulin-coated plates.

A cell separation method using immunoglobulin (Ig)-coated plates, originally devised for murine spleen cells, was modified and adapted for enrichment (and depletion) of cellular subpopulations from human peripheral blood. For the direct separation of B and T cells, F(ab')2 fragments of anti-human Ig were used to coat the plates. For indirect separation, the cells were first incubated with monoclonal antibodies to cell surface antigens and then separated in plates coated with anti-mouse Ig. Plates were first coated with poly-L-lysine to facilitate the adherence of anti-Ig antibodies, and finally with bovine serum albumin to mask free poly-L-lysine. Cells which did not react with the anti-Ig antibodies or which were nonadherent to the plate were pipetted off; cells which reacted with the anti-Ig antibodies or which were adherent were eluted after incubation with excess serum. T, non-T, T4+, T4-, T8+, and T8- lymphocytes were separated with high viability, purity, and yield. The method was used to study suppressor activity of a patient who was treated by bone marrow transplantation for myelofibrosis. Strong suppressor activity was associated with unfractionated peripheral blood mononuclear leukocytes, monocytes, T, T8+, and T4- cells but not with B, T8-, and T4+ cells.

Antibodies, Monoclonal↗
PubMed1982

Isolated phosphorylcholine binding lymphocytes. I. Use of a cleavable crosslinking reagent for solid-phase adsorbent isolation of functional antigen binding cells.

Isolation and characterization of BALB/c lymphocytes specific for phosphorylcholine is described. The isolation protocol utilizes phosphorylcholine coupled to gelatin coated plates via the cleavable crosslinking reagent N-succinimidyl 3-(2-pyridyldiothio)propionate (SPDP). The procedure is rapid, requiring only 1-2 h and conducted entirely at 0-4 degrees C. Hapten binding cells are eluted by vigorous pipetting at 4 degrees C with medium containing 20% fetal calf serum. Approximately 70% of isolated cells rebind antigen as assessed using a PC Brucella abortus rosette assay while 50% express the TEPC15 idiotype. Approximately 85% of isolated and idiotype positive cells are B cells while the remainder are T cells. Limiting dilution analysis revealed that approximately 1/5 of PC binding cells isolated from the spleens of normal mice respond to lipopolysaccharide plus dextran sulfate by production of anti-PC antibody. Approximately 1/11 respond to PC Brucella abortus and 1/250 respond to PC sheep erythrocytes plus primed T cells by anti-PC antibody production. The results clearly demonstrate the effectiveness of this technique for isolation of highly enriched, functional, antigen specific lymphocytes.

Animals↗
PubMed1989

A quantitative C-reactive protein assay using latex agglutination in microtiter plates.

In this quantitative assay, latex particles coated with anti-C-reactive protein (CRP) are agglutinated by CRP following vortex agitation in microtiter plates. A decrease in absorbance at 405 nm is directly proportional to CRP concentration. This 30 min assay is simple and necessitates only two pipetting steps after serum dilution. The linear part of the standard curve ranges from 5 to 150 mg/l and CRP concentrations up to 300 mg/l can be determined without additional dilution of sera. Within-assay reproducibility varies from 4.4% to 7.5% while between-assay reproducibility ranges from 9.3% to 12.2%. Correlation studies performed with 104 sera assayed by automated nephelometer and turbidimeter gave correlation coefficients of 0.96 and 0.97 respectively.

C-Reactive Protein↗
PubMed1991

Immunoturbidimetric assay of beta 2 microglobulin using latex particles in microplates.

A latex particle immunoturbidimetric assay in microplates has been developed for the quantitation of beta 2 microglobulin. The assay which involves three pipetting steps and one incubation, has a clinically useful range of 0.4-16 mg/l. A comparison of the method with a commercial radioimmunoassay in the analysis of 125 sera revealed a high degree of correlation. Although the assay was performed manually, it showed considerable potential for full automation.

Agglutination Tests↗
PubMed1990

Electrophoretic characterization of chromosomal DNA from two microsporidia.

Spores of two microsporidia, Nosema pyrausta (from the European corn borer, Ostrinia nubilalis) and N. furnacalis (from the Asian corn borer, O. furnacalis) were harvested from laboratory-reared O. nubilalis caterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different. N. pyrausta spores were incubated in 0.1 N KOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37 degrees C to induce germination. N. furnacalis spores were first washed in 10 mM Na2EDTA in 1 mM Tris base, pH 7.5, exposed to 0.01 N KOH in 0.17 M KCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17 M KCl in 10 mM Na2EDTA (pH 8), at 37 degrees C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50 degrees C in 10 mM Tris base/100 mM Na2EDTA, pH 7.8, with 1 mg/ml proteinase K and 1% N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs in N. pyrausta and N. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.

Animals↗
PubMed1983

Doublet formation of diabetic erythrocytes as a model of impaired membrane viscous deformation.

Erythrocyte deformation involves both viscous dissipation in the cell interior and viscoelastic motion of the cell membrane. Reports that describe reduced filterability of diabetic erythrocytes, altered response to oscillatory motion in a capillary-sized pipet, and impaired packing during centrifugation indicate a disturbance of red cell rheology in diabetes. We have selected conditions that minimize the macromolecule-mediated energy of attraction between erythrocytes and studied erythrocyte motion during doublet formation. Under such conditions, doublet formation frequency is strikingly reduced in diabetes. For nondiabetic erythrocytes the formation rate is 0.73 doublets per minute, whereas for diabetic erythrocytes the rate is 0.23 doublets per minute. In addition, mean velocity of doublet formation was found to be decreased to half of normal in diabetes. Completeness of doublet formation, regularly diminished when cell size of the two component cells was similar, was the same for diabetic and nondiabetic erythrocytes. Observation of several features of doublet formation gave a picture of the mechanical process. The initial cell making contact with the glass microscope slide was observed to remain fixed in position. The late arriving cell's ability to form a doublet was seen to decrease rapidly, apparently because it came to adhere to the glass surface. The attractive force between the cells overcomes the force of gravity, but cell deformation resistance slows doublet formation by balancing the tendency for cell-cell contact area to increase. An integral equation combining strain energy and viscous dissipation was applied to the doublet formation process. Slowing of doublet formation in diabetes appears to be produced by a doubling of resistance to rate of change of curvature of diabetic erythrocytes.

Diabetes Mellitus↗
PubMed1992

Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo.

Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.

Animals↗