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At least 217 records · Page 12Linked to original sources
PubMed1994

The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells.

Mechanical stimulation of one mammary tumor cell in culture induced an increase in its intracellular calcium concentration which spread to surrounding cells. The increase in calcium can also be induced by addition of a solution in which cultured mammary tumor cells were stimulated by repeated pipetting (solution after pipetting cells, SAPC). The activity of the SAPC was completely abolished by treatment with snake venom phosphodiesterase or pyrophosphatase. Uridine triphosphate (UTP), uridine diphosphate (UDP) and ATP (1 microM each) were detected in the SAPC, whereas 5'-UMP and 5'-AMP were produced by phosphodiesterase digestion. A mixture of UTP, UDP and ATP (1 microM each) elicited a calcium response which was comparable to that induced by SAPC, while UTP, UDP or ATP alone at 1 microM elicited a small increase in calcium concentration in mammary tumor cells. Suramin, a competitive antagonist of P2 purinoceptors, diminished the spreading of the calcium wave induced by mechanical stimulation. It also blocked the responses to SAPC, UTP, UDP and ATP. These findings suggest that the mechanical stimulation results in the release of UTP, UDP and ATP into the extracellular space which mediates induction of the spreading calcium response via P2U-type purinoceptors.

Adenosine Triphosphate↗
PubMed1979

The effects of pressure on the water permeability of the descending limb of Henle's loops of rabbits.

Descending limbs of Henle's loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional "Sylgard seal" pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H2O) even when the bath was hyperosmotic: delta Jv = -0.06 +/- 0.18 nl/min (n = 7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16 +/- 8 mM (n - 7) and 254 +/- 38 mOsm (n = 7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea. When the "Sylgard seal" collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H2O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 +/- 5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153 +/- 8 mmole/l; 6 tubules). The 14C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 x 10(-7) cm2 . s-1 +/- 0.23 x 10(-7) (11 tubules). When the bath was made hyperosmotic using urea or raffinose the 14C urea permeability increased significantly.

Animals↗
PubMed1989

Isolation of Mycoplasma bovis from intact and microinjected preimplantation bovine embryos washed or treated with trypsin or antibiotics.

Incubation of day 7 bovine embryos with 10(4) or 10(6) CFU/ml of Mycoplasma bovis (M. bovis) or microinjection of M. bovis into the cells of day 7 embryos did not influence embryonic development. M. bovis was recovered from all embryos washed 10 times by a standard pipetting method or vortexed and pipeted 10 times. M. bovis was also recovered from zonae pellucidae removed and washed from microinjected embryos. Neither treatment with trypsin nor exposure of embryos to combinations of penicillin, streptomycin, lincomycin and spectinomycin, or gentamicin, tylosin, lincomycin, and spectinomycin, inactivated M. bovis.

Animals↗
PubMed1983

Improved method for processing autoradiographs of cells grown on multiwell plates.

A modification of an established procedure for autoradiographic processing of cultured cells is described. This method eliminates the need for pipetting each individual well and also for cutting and dismantling the multiwell plate for slide preparation. In this procedure the entire plate can be processed as a single unit and the cells can be analyzed in situ, thus eliminating the time consuming pipetting and cutting procedures. Furthermore, the entire experiment can be filed without use of additional slides or storage boxes. Hence, this is a simpler, time conserving, and economical way to process large numbers of cultures for thymidine labeling indices.

Animals↗
PubMed1998

Quantifying the cleanliness of glass capillaries.

I used capillary rise methods to investigate the lumenal surface properties of quartz (fused silica, Amersil T-08), borosilicate (Corning 7800), and high-lead glass (Corning 0010) capillaries commonly used to make patch pipets. I calculated the capillary rise and contact angle for water and methanol from weight measurements. The capillary rise was compared with the theoretical maximum value calculated by assuming each fluid perfectly wetted the lumenal surface of the glass (i.e., zero contact angle, which reflects the absence of surface contamination). For borosilicate, high-lead, and quartz capillaries, the rise for water was substantially less than the theoretical maximum rise. Exposure of the borosilicate, lead, and quartz capillaries to several cleaning methods resulted in substantially better--but not perfect--agreement between the theoretical maximum rise and calculated capillary rise. By contrast, the capillary rise for methanol was almost identical in untreated and cleaned capillaries, but less than its theoretical maximum rise. The residual discrepancy between the observed and theoretical rise for water could not be improved on by trying a variety of cleaning procedures, but some cleaning methods were superior to others. The water solubility of the surface contaminants, deduced from the effectiveness of repeated rinsing, was different for each of the three types of capillaries examined: Corning 7800 > quartz > Corning 0010. A surface film was also detected in quatz tubing with an internal filament. I conclude that these borosilicate, quartz, and high-lead glass capillaries have a film on the lumenal surface, which can be removed using appropriate cleaning methods. The surface contaminants may be unique to each type of capillary and may also be hydrophobic. Two simple methods are presented to quantitate the cleanliness of glass capillary tubing commonly used to make pipets for studies of biological membranes. It is not known if the surface film is of importance in electrophysiological studies of biological membranes.

Capillary Action↗
PubMed2000

Development of a rapid and sensitive bioassay device using human cells immobilized in macroporous microcarriers for the on-site evaluation of environmental waters.

We developed a novel disposable bioassay device based on the fluorescein isothiocyanate-labelled low-density lipoprotein-uptake activity of human hepatoblastoma Hep G2 cells. The cells were cultured in porous microcarriers at a high cell density and packed in a filter tip that has a hydrophobic membrane. Upon evaluation of water samples, the culture medium was decanted by pipetting it down with a micropipet, and the samples were then introduced to the cell-immobilizing part of the tip only by pipetting them up after mixing them with x10 concentrated culture medium. The new device enabled us to detect almost the same toxicity levels of river water within 2 h of exposure as those detected by a conventional 48-h cell-survival assay. This is the first bioassay device for the rapid on-site evaluation of environmental waters using cultured human cells, and therefore promising for water-quality management based on risk to humans.

Biological Assay↗
PubMed1987

A microtiter plate assay for protein kinase C.

We report the development of a microtiter plate assay for protein kinase C. Reaction components and enzyme samples (protein kinase C purified by phosphatidylserine/cholesterol affinity or DEAE-Sephacel ion-exchange chromatography) were added to wells of a 96-well microtiter plate. The assay was started by the addition of [gamma-32P]ATP with a repeating pipet. After a 3-min incubation at 30 degrees C the wells were sampled six at a time with a 12-channel pipet and spotted onto phosphocellulose filter paper rectangles which were washed with tap water and acetone and counted for radioactivity. The microtiter plate method was more rapid than but gave results similar to those of a standard assay performed in plastic test tubes individually incubated in a 30 degrees C water bath. The microtiter plate procedure gave an intraassay (within one plate) variation of less than 9% and an interassay (between plates) variation of less than 5%. It was linear with time of incubation for 20 min and with amount of enzyme. This method can be used to expedite the assaying of column chromatography fractions for protein kinase C (and other kinase) activity.

Cholesterol↗
PubMed1988

Multiple uses of liquepipets in Southern, northern, and western blots.

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.

Blotting, Northern↗
PubMed1985

Disposable microliter immunoabsorbent columns: construction and operation.

Disposable microliter immunoabsorbent columns were constructed from pasteur pipets. The bed support was a cube of gelatin surgical sponge, which was tamped into the pipet tip. Column dead space, represented by the compressed volume of the sponge, was 5 microliters. The columns were used with protein A-Sepharose; settled bed volumes were 50 microliters. It was possible to pour columns that functioned as immunoabsorbents with bed volumes as small as 10 microliters. There was no gravity flow through these columns. Flow was achieved by touching column tips to absorbent paper if liquid was to be discarded or to 50 microliters capillary tubes for fluid collection. A simple capillary collection tube assembly was designed for operation of a row of 10 columns at a time. In a test system of [3H]methotrexate and IgG anti-methotrexate, 90% of applied antigen was bound to antibody columns, whereas 90% was recovered in the eluates from control columns. The columns were used in the initial step of screening uncloned hybridoma culture fluids for anti-MSP.

Animals↗
PubMed1986

Personal computer-controlled microsurgery of fertilized eggs and early embryos.

The microsurgery of mouse and rat eggs and early embryos was attempted using a micromanipulator driven by three pulse motors. The pulse signals that regulate the three pulse motors for the X, Y, and Z axes were controlled according to the personal computer programs produced on the basis of the displayed data. As a result, the following was found. 1) The computer-controlled operation was possible in the X and Y plane on a specimen previously suctioned and retained by a holding pipet. A microinjection pipet was inserted into the male pronucleus of a fertilized egg and the morula was bisected using a microblade; these microtools were moved horizontally. 2) A more complicated micromanipulation in two dimensions (X and Z axes), which is very difficult manually, was possible by using this system. 3) microsurgery (microinjection of a fluorescent material (FITC) into the male pronucleus, enucleation of a fertilized egg, and vertical or horizontal bisection of morulae) was carried out successfully by a student who had no practical experience in this field. These facts suggest that the system markedly facilitates microsurgery, without need for full training in the manual procedures.

Journal Article↗
PubMed1992

Diagnostic techniques for transvaginal-transuterine aspiration of bovine fetal fluid during the early fetal period.

Studies were designed to evaluate 2 methods for transvaginal-transuterine collection of bovine fetal fluids. The first technique (direct) required simultaneous transrectal palpation and retraction of the gravid uterus and direct, intravaginal manipulation of a needle and vacuum tube assembly. The direct technique was only suitable for use in multiparous animals and was attempted when fetal age ranged from Day 55 to Day 75. The second technique (indirect) may be used in primiparous cows, because aspiration was accomplished through a plastic infusion pipet, altered by attachment of a needle to its tip. When this technique was used, fetal age ranged from Day 50 to Day 65. The direct technique provided more control over needle placement and resulted in a higher success rate for aspiration of fetal fluid following single needle penetration (77 versus 50%). Both techniques were associated with rates of abortion (3/13 for the direct and 4/10 for the indirect) that were judged to preclude prospective use in diagnostic strategies for first trimester fetal wastage. Within the controlled study, the diagnostic quality of the aspirate was determined. It was concluded that the altered pipet technique provided aspirates that were of diagnostic, noncontaminated quality. Field use of fetal fluid aspiration following discovery of nonviable pregnancies by B-mode ultrasonography is discussed.

Journal Article↗
PubMed1992

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes.

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.

Journal Article↗
PubMed1989

Separation of B and T lymphocytes by cellular adsorption chromatography with polyamine graft copolymers as column matrices. II. Recovery of adsorbed B cell enriched populations from the column.

Poly(2-hydroxyethyl methacrylate)-graft-polyamine copolymers were developed for column adsorbents for separating lymphocyte subpopulations collected from rat mesenteric lymph nodes. Bead-shaped adsorbents were prepared by coating poly(2-hydroxyethyl methacrylate)-graft-polyamine copolymer on glass beads. Separation features of the column packed with poly(2-hydroxyethyl methacrylate)-graft-polyamine copolymer-coated glass beads were evaluated under various operating conditions. Separation efficacy, AB/AT, was significantly affected by the infusion rate of lymphocyte suspension into the column, increasing with decreasing infusion rate. T cell purity in the column effluent was almost 95% at the infusion rate of 0.1 ml/min. The infusion rate was also found to affect the detachment of adsorbed lymphocytes from the poly(2-hydroxyethyl methacrylate)-graft-polyamine copolymer surface by mechanical pipetting; lymphocytes adsorbed at a low infusion rate were able to be detached quantitatively by suspending the lymphocyte-adsorbed beads with gentle pipetting. The detached lymphocytes thus obtained were confirmed to be enriched in B cells 1.4 times the initial mixture of B and T cells. The addition of albumin in the medium was found to affect the processes of adsorption and recovery of lymphocyte.

Adsorption↗
PubMed1987

Bubble pressure measurement of micropipet tip outer diameter.

The bubble pressure of a micropipet tip is the minimum transmural pressure necessary to expel bubbles into a liquid in which the tip is submerged. Bubble pressure measurement is a simple, accurate and non-destructive technique for determining the tip outer diameter of micropipets used in microinjection, intracellular recording and patch-clamp experiments. For micropipets pulled from thin-wall glass capillary tubing, with tip outer diameters between 0.1 and 1.5 micrograms, observed bubble pressures are close to those predicted by considering surface tension at the pipet tip. Also, as predicted, neither glass composition nor shank geometry influences the relationship between bubble pressure and tip outer diameter. Tip outer diameter for a given bubble pressure is larger for micropipets pulled from thick-wall glass capillary tubing than it is for pipets pulled from thin-wall glass. However, for a given wall thickness, the outer diameter vs bubble pressure behavior is sufficiently reproducible to allow accurate size determination from bubble pressure measurements. The size of micropipet tips modified by fire-polishing or beveling can also be estimated by bubble pressure measurement, although with less accuracy. Thus, bubble pressure measurement can be a useful technique for following the progress of micropipet tip modification.

Calibration↗
PubMed1992

A protocol for rapid screening of proteoglycan-degrading metalloproteinase inhibitors.

A procedure is described for rapid, high-volume screening of proteoglycan-degrading metalloproteinase inhibitors. The procedure was developed by modifying the proteoglycan-polyacrylamide particle assay for proteases, polysaccharidases (Nagase and Woessner, 1980), and the 1,9-dimethylmethylene blue (DMB) assay for sulfated glycosaminoglycans (Farndale et al., 1982). The following modifications have made the new protocol suitable for evaluating a large number of compounds simultaneously: 1) Pipetting steps were automated using a pipetting robot. 2) Speed and sensitivity of the 1,9-dimethylmethylene assay were increased by scaling down the assay and measuring absorbance using an automatic multiwell plate reader. 3) Data capture and analysis were simplified using computer programs. The modified procedure is rapid, sensitive, reliable, and requires a small sample size. Above all, the procedure is suitable for high-volume screening: approximately 100 compounds can be evaluated in 1 day.

Acrylic Resins↗
PubMed2006

Real-time GUS analysis using Q-PCR instrumentation.

The development of new technology within biological sciences has resulted in a number of real-time PCR instruments that have become essential tools within molecular biology. This equipment has facilitated high throughput analysis of samples and optimal information gathering of completed PCR reactions for example estimating the copy number of a gene of interest that is inserted into particular genomes. Real-time PCR instruments frequently come with optional filter sets, e.g. the ALEXA filter set which has parameters in common with excitation and emission wavelengths of sodium methyl umbelliferone (NaMU) widely used in beta-glucuronidase reporter gene assays. Using these filter sets it has been possible to quantify and measure gus A activity of Ulmus procera SR4 in real-time removing the necessity for aliquots of reactions to be stopped by pipetting into carbonate buffer for each time point. The introduction of real-time GUS analysis leads to faster, more accurate and reproducible assays with reduced potential for pipetting errors, requires fewer manipulations and encourages high throughput analysis of inter-individual gene expression variation.

Equipment Design↗
PubMed2006

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria.

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.

Animals↗
PubMed2001

Aspiration of human neutrophils: effects of shear thinning and cortical dissipation.

It is generally accepted that the human neutrophil can be mechanically represented as a droplet of polymeric fluid enclosed by some sort of thin slippery viscoelastic cortex. Many questions remain however about the detailed rheology and chemistry of the interior fluid and the cortex. To address these quantitative issues, we have used a finite element method to simulate the dynamics of neutrophils during micropipet aspiration using various plausible assumptions. The results were then systematically compared with aspiration experiments conducted at eight different combinations of pipet size and pressure. Models in which the cytoplasm was represented by a simple Newtonian fluid (i.e., models without shear thinning) were grossly incapable of accounting for the effects of pressure on the general time scale of neutrophil aspiration. Likewise, models in which the cortex was purely elastic (i.e., models without surface viscosity) were unable to explain the effects of pipet size on the general aspiration rate. Such models also failed to explain the rapid acceleration of the aspiration rate during the final phase of aspiration nor could they account for the geometry of the neutrophil during various phases of aspiration. Thus, our results indicate that a minimal mechanical model of the neutrophil needs to incorporate both shear thinning and surface viscosity to remain valid over a reasonable range of conditions. At low shear rates, the surface dilatation viscosity of the neutrophil was found to be on the order of 100 poise-cm, whereas the viscosity of the interior cytoplasm was on the order of 1000 poise. Both the surface viscosity and the interior viscosity seem to decrease in a similar fashion when the shear rate exceeds approximately 0.05 s(-1). Unfortunately, even models with both surface viscosity and shear thinning studied are still not sufficient to fully explain all the features of neutrophil aspiration. In particular, the very high rate of aspiration during the initial moments after ramping of pressure remains mysterious.

Cytoplasm↗