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 433 records · Page 24Linked to original sources
PubMed1985

Evaluation of a method to study the uptake of prednisolone sodium phosphate from different oral mucosal sites.

A filter paper disk method for the delivery of drugs to oral mucosal sites was evaluated. The water-soluble steroid prednisolone sodium phosphate was chosen as the tracer drug. The in vitro recovery of the steroid from the filter paper disks after placement onto glass was greater than 95% and within the pipetting error for the method. Filter paper disks containing 1.25 mg doses of prednisolone sodium phosphate were placed on three different intraoral sites for 5 minutes and the drug retention was calculated in six volunteers. The mean percentage retention at the three sites was 65.4% for the sublingual mucosa, 24.1% for the buccal mucosa, and 9.9% for the palatal mucosa. The differences between the sites were all significant. The method may be useful for studying the retention of other drugs at various oral mucosal sites and for determining the factors that affect such retention.

Absorption↗
PubMed1982

A rapid radioimmunoassay, using a nylon support, for amatoxins from Amanita mushrooms.

A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.

Agaricales↗
PubMed1986

A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte.

Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min.

Animals↗
PubMed1989

Trace element profiles in oesophageal neoplasms determined by proton-induced X-ray emission.

Trace element concentrations were determined in surgically resected, cancerous human oesophageal tissue by proton-induced X-ray emission (PIXE) analysis. Each sample was divided into six zones, E3 and E4 formed the central tumour region, E1 and E2 were proximal to the oesophagus and E5 and E6 distal to the oesophagus. Malignancy was confirmed histopathologically in all cases. Samples were dissolved in NaOH solution and pipetted onto a thin mylar film for analysis by a proton beam from a Van de Graaff accelerator. Titanium, Fe, Cu, Zn, As, Rb, Br and Pb were detected in varying concentrations. Of these, Ti, Fe, Cu, Zn and As showed specific behaviour in the six zones.

Carcinoma↗
PubMed1976

The effects of topical saline and isoproterenol on nasal airway resistance.

Following determination of baseline nasal airway resistance (NAR) in human subjects, phosphate-buffered saline (PBS) was aerosolized into each nostril and NAR measurements repeated 8 times in 15 minutes. The mean maximal NAR increase in 102 subjects was 22.5% (SD +/- 24.5%) with no significant difference between atopic and nonatopic persons. Hay fever patients did not show significantly increased responsiveness to PBS while symptomatic. During 242 repeat challenges with PBS in 67 asymptomatic subjects, NAR increased over 50% in 24 tests. Following a series of 6 PBS challenges at 15-minute intervals, 10 of 71 subjects had more than a 100% increase in NAR over initial baseline values. Administration of PBS on cotton pledgets, pipetting PBS into the nose, or even just inserting a nasal speculum produced greater increases in NAR than the usual aerosol method. Intranasal atropine effectively blocked PBS-induced increases in NAR, suggesting parasympathetic stimulation as a mechanism. Isoproterenol increased NAR over 100% in 27 of 53 subjects, with no significant difference among rhinitic, asthmatic, and nonatopic individuals. This effect was inhibited by propranolol. Isoproterenol administration to 12 ragweed-sensitive subjects 15 minutes prior to ragweed challenge produced a variable inhibition in NAR responses or no protection at all. Thus the direct effects of isoproterenol on the nasal vasculature tend to outweigh those expected from inhibition of mediator release under the conditions of this study.

Administration, Intranasal↗
PubMed1989

Nasal histamine challenges in symptomatic allergic rhinitis.

Twenty subjects (seven with perennial allergic rhinitis, seven with symptomatic seasonal allergic rhinitis, and six normal control subjects) underwent assessment of nasal sensitivity to histamine. Nasal resistance was measured by posterior rhinometry under control conditions and after log incremental doses of histamine solution pipetted into the nose (0.5 to 5000 micrograms). Allergic subjects exhibited a twofold rise of nasal resistance with doses of 0.5, 5, or 50 micrograms of histamine, whereas the nasal resistance in normal subjects remained unchanged until 500 or 5000 micrograms of histamine had been administered. Nasal reactivity to histamine was not correlated with symptoms on the day of testing but was correlated with the number of positive wheals to skin prick testing. It was concluded that nasal resistance is more sensitive to histamine in subjects with allergic rhinitis than in normal control subjects and that this difference may be used as the basis of a diagnostic test.

Administration, Intranasal↗
PubMed1992

Lack of antibody specificity by mouse trophectoderm during immunosurgery.

Immunosurgery of blastocyst-stage embryos is an effective procedure for isolating the inner cell mass. The ability of rabbit sera antibodies produced to interspecific types of cells to bind to mouse trophectoderm antigens and mediate complement-reactive lysis was investigated. Fifty hatched and 25 expanded blastocysts per treatment were exposed to 1 of 7 heat-inactivated polyclonal antibodies (1: 10 DMEM dilution) produced in rabbits to mouse brain cells (MBr), mouse lymphocytes (MLy), rat lymphocytes (RLy), hamster lymphocytes (HLy), cattle splenocytes (CSp), sheep splenocytes (SSp), and pig splenocytes (PSp). After subsequent incubation in a guinea pig complement solution (1: 16 dilution) the embryos were assessed for trophectoderm lysis, and the inner cell masses were pipetted free of cellular debris. Fewer (P<0.01) hatched blastocysts were lysed completely when treated with RLy (60%), CSp (50%) and PSp (14%) antibodies compared the other treatment groups (100%). A similar response was observed with expanded blastocysts, with the exception that even fewer (P<0.01; RLy, 24%; CSp, 40%; PSp, 0%) were lysed completely. Forty percent of the PSp expanded blastocysts experienced no lysis, which was different (P<0.01) than in all the other treatments. Secondary experiments were performed to explain the cause of partial lytic response. Overall, the results indicate that interspecific antibodies can bind to murine trophectoderm surface antigens and mediate immunosurgical inner cell mass isolation, although the trophectoderm lytic response varied with antibody source.

Journal Article↗
PubMed1995

PCR-sexing of bovine embryos: a simplified protocol.

To make bovine embryo sexing under farm conditions more feasible we developed a simplified protocol utilizing manual biopsy and detection of the Y chromosome directly from polymerase chain reaction (PCR) reaction tubes. Twenty-four embryos (morulae and blastocysts) were biopsied manually into 2 to 4 samples. One sample of each original embryo was diagnosed for sex, based on restriction fragment length polymorphism of PCR-amplified DNA of the ZFX/ZFY locus. The remaining 44 samples were diagnosed using the tube detection assay. In this assay the biopsies were pipetted into 0.5 -ml reaction tubes containing lysis mixture, incubated 10 to 60 min at 37 degrees C and inactivated 10 min at 98 degrees C. Then the PCR mixture was added containing buffer, DNA polymerase, ethidium bromide and primers designed to amplify the highly repeated btDYZ-1 region of the bovine Y chromosome. After 50 cycles of PCR, the reaction tubes were examined under UV illumination for pink fluorescence indicating the presence of Y-chromosomal DNA. All sexing results from the replicates were in agreement with the ZFX/ZFY assay, with 12 of the original embryos diagnosed as females and 12 as males. We conclude that highly efficient and accurate PCR-sexing of embryos can be accomplished without the use of micromanipulators, control primers and electrophoresis. The 2 reaction mixtures needed for sex diagnosis can be stored at -20 degrees C and -196 degrees C, respectively. The tube detection assay minimizes the risk of carryover contamination by previously amplified products as there is no need to open the tubes following PCR.

Journal Article↗
PubMed1988

Immunoaffinity chromatography of lymphocyte subpopulations using tert-amine derived matrices with adsorbed antibodies.

New polymeric solid-phase matrices for cell affinity chromatography were prepared and their advantageous characteristics compared with conventional matrices were highlighted. These new matrices are derivatives of poly(2-hydroxyethyl methacrylate) (PHEMA) containing a slight quantity of amino compounds as a co-monomer. They were applied to immunoaffinity selection between IgG+ and IgG- lymphocytes of the rat mesenteric lymph node. Simple physical adsorption was sufficient for anti-rat IgG antibodies to be immobilized on these matrices, allowing us to omit the laborious procedure of covalent-linking of antibodies on a matrix. As these matrices themselves showed extremely low non-specific adsorption of lymphocytes, a very dilute solution of antibody (0.02-0.08 mg/ml) was enough for column conditioning. This separation method gave IgG- lymphocytes of more than 90% purity and almost 95% yield within as short a time as 7 min. Further, IgG+ lymphocytes were obtained in good yield (80-90% of loaded number) by recovering the adsorbing cell fraction from the column by gentle pipetting of the matrix.

Amines↗
PubMed1982

A simple method for preparing single cell suspensions of heart and smooth muscle for radioreceptor labeling studies.

A simple method of preparing single cell suspensions for radioligand binding studies is described. The method involves incubating tissues in the presence of collagenase and elastase for 90 min in physiological solution with 1 mM calcium chloride and the mechanical disruption of the tissue by pipetting. The tissues examined were atria, ventricle, bladder, uterus, and taenia coli removed from mature guinea pigs. Viability of the cells by trypan blue exclusion showed 60-88% viable cells and receptor binding studies using (3H]-1-quinuclidinyl benzilate (QNB) yield KD values of approximately equal to 0.1 nM. The receptor numbers for each tissue were (receptors/cell): atria, 5700; ventricle, 11,000; uterus, 31,000; bladder, 44,000; taenia coli, 68,000.

Animals↗
PubMed1983

Use of an extracorporeal arterio-venous shunt and capillary tubes for frequent micro-blood sampling in rats.

Because of the importance of frequent sampling during kinetic analysis, we have developed a method that permits frequent, reliable, and rapid access to the blood stream in rats. The method comprises the establishment of an extracorporeal arterio-venous shunt between femoral blood vessels. Quantitative samples of whole blood and plasma are obtained with capillary micro hematocrit tubes. The small sample volume permits repetitive analysis without major changes in hematocrit. There is good agreement between these measures and ordinary pipetting techniques. Kinetic analysis showed good resolution with close correspondence between actual measurements and the final curve of best fit. This method could be employed advantageously in small conscious animals, when frequent small samples are required by experimental design.

Animals↗
PubMed1986

In vitro evaluation of nucleoside transport inhibition in vivo.

A method is described which permits rapid evaluation of the effects of nucleoside transport inhibitors on [3H]thymidine uptake into mouse whole blood cells. The uptake is measured after 15-20 sec by withdrawing 100 microliters of blood from the orbital sinus into microcapillary pipets that have been prelabeled with [3H]thymidine. Blood cells are rapidly separated from plasma by centrifugation through a layer of dibutyl phthalate using an Eppendorf microcentrifuge. The acid-soluble radio-activity associated with the cell pellet is then determined. Treatment of mice with the nucleoside transport inhibitors dipyridamole or p-nitrobenzylthioinosine 5'-monophosphate reduces uptake of the radioisotope into the cells. The assay allows for estimation of the dose- and time-effect relationships for inhibition of nucleoside transport by these agents. Advantages of the method are its simplicity, its speed, and the fact that the biological sample and drug are not diluted before measurement of the drug effect.

Animals↗
PubMed1985

Modification of the vitamin K-dependent carboxylase assay.

Methods are presented that describe alternative protocols for the isolation of rat liver microsomes containing the vitamin K-dependent carboxylase and the procedure in which the solubilized enzyme is assayed. The method for determining the rate of 14CO2 incorporation into low molecular weight, acid soluble substrates by the rat liver microsomal vitamin K-dependent carboxylase has been modified in order to optimize safety, accuracy and simplicity. For these studies the rat liver microsomes containing the vitamin K-dependent carboxylase were isolated by CaCl2 precipitation. These Triton X-100 solubilized microsomes were found to be equivalent to the microsomes obtained by high speed ultracentrifugation with regard to protein concentration, pentapeptide carboxylase activity, carboxylase activity, preprothrombin concentration and total carboxylatable endogenous protein substrate. This modified assay procedure requires fewer steps and pipetting transfers and is quantitatively equivalent to previously employed protocols. The described technique can be adapted for any assay where 14CO2 or H14CO3- is incorporated into non-volatile products. This newly developed assay procedure was employed to assess conditions necessary for optimal vitamin K-dependent carboxylation of the less expensive substrate, N-t-Boc-L-glutamic acid alpha-benzyl ester. The optimal conditions for the carboxylation of N-t-Boc-L-glutamic acid alpha-benzyl ester by the carboxylase were found to be 10 mM N-t-Boc-L-glutamic acid alpha-benzyl ester, 10 mM MgCl2 at 15-18 degrees C. The rate of N-t-Boc-L-glutamic acid alpha-benzyl ester carboxylation under these optimized conditions was found to be higher (1.5-fold) than the rate of carboxylation of 1 mM Phe-Leu-Glu-Glu-Ile in the presence of the cation activator, MgCl2.

Animals↗
PubMed1986

Determination of protein by Coomassie dye-binding in agarose gels.

A variation of the Coomassie dye-binding assay for proteins is described. Protein samples were pipetted to the surface of agarose plates in uniformly sized spots and stained with Coomassie Blue G-250. The bound dye was determined by densitometric scanning using double wavelength and flying spot facilities. The response curves were linear in an about 10-fold concentration range with a lower detection limit of 0.5 microgram. No background correction was necessary because unbound dye and most substances known to interfere with other protein assays were removed during the staining and destaining of the agarose gels. Membrane proteins could be analyzed since the samples were applied as solutions in 1% sodium dodecyl sulfate.

Animals↗
PubMed1987

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay.

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.

Aprotinin↗
PubMed1987

Quantitative electroelution of oligonucleotides and large DNA fragments from gels and purification by electrodialysis.

We have designed a new device [Biotrap (Elutrap in the U.S.A. and Canada), available from Schleicher & Schuell] for electroelution, -concentration , and -dialysis of DNA and other charged macromolecules above 5 kDa. In an electric field, the DNA migrates in an open channel out of the gel slice through a microporous membrane, BT2, into the trap section, where it is retained by a very dense, non-adsorbant, and inert membrane BT1. Specifically designed for use in an electric field, the matrix of this new membrane is much denser than that of dialysis membranes. In contrast to dialysis membranes, BT1 will not adsorb large DNA fragments nor allow passage of small DNA fragments when subjected to an electric field. In the absence of an electric field, BT1 and BT2 effectively seal the trap, maintaining the final elution volume of the purified sample. The trap can contain from 200-600 microliter and is collected from above with a pipet. In the experiments described here, 85-95% of oligonucleotides (14-mer) and large (150 kb) DNA fragments were recovered, independent of fragment length. The purity of the eluted DNA was demonstrated by restriction enzyme digestion, nick-translation, primer extension, end-labeling with polynucleotide kinase, and ligation. Electrodialysis was successfully used for the complete removal of common contaminants inhibiting the polynucleotide kinase reaction and for the removal of CsCl from DNA samples.

DNA↗
PubMed1992

Simultaneous recording of [Ca2+]i increases in isolated olfactory receptor neurons retaining their original spatial relationship in intact tissue.

A new method is described for isolating olfactory receptor neurons suitable for simultaneous recording of odorant responses in several cells. This method, called "tissue printing" by Cassab and Varner, was used to isolate cells for measurement of odorant-induced increases in cytosolic-free calcium concentration ([Ca2+]i) using the Ca2+ indicator dye fura-2. A large number of receptors could be isolated from a piece of olfactory epithelium (about 300 microns square), preserving their normal morphology and relative local topology to that in the intact olfactory tissue. The probability that there are one or more receptor cells with odorant-induced responses in [Ca2+]i per preparation was 4 times higher with cells isolated by the tissue printing than with those obtained by the pipetting method. The responses of 2 receptor cells separated by 28 microns in the recording chamber differed for 2 odorants: isoamyl acetate and citralva. The method was useful for isolating receptor neurons without losing their morphological features and for investigating the spatial distribution of odorant responsiveness of each receptor over the olfactory epithelium.

Animals↗
PubMed1994

An alternative method for obtaining high-viability cell suspensions from neonatal mouse brain.

Cultured brain cells have contributed greatly to our understanding of a variety of neurobiological processes. The ability to culture brain tissue is important for studying cellular processes underlying unique neural properties. Traditional culturing techniques commonly involve triturating tissue through glass Pastuer pipets, which are inappropriate for use with potentially biohazardous materials. We therefore developed an alternative method for dissociating brain tissue. The protocol combines enzymatic digestion and mechanical dissociation with additives to the dissection medium that protect the cells against other sources of injury, including glutamate neurotoxicity, oxidative damage, and excessively alkaline pH. We find this method works well with post-natal mouse brain, consistently giving cell viabilities in the range of 92-99% and an average yield of 3.1 x 10(6) cells per mouse.

Animals↗