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At least 361 records · Page 20Linked to original sources
PubMed1993

Trophectoderm microbiopsy in murine blastocysts: comparison of four methods.

PURPOSE: This study compares four trophectoderm microbiopsy techniques for removal of blastomeres from murine blastocysts: (1) aspiration, trophectoderm pipetted through the zona; (2) incision, trophectoderm excised with a microrazor; (3) slit/excision, the zona slit and herniating trophectoderm excised; and (4) hatch/excision, trophectoderm cells excised after spontaneous hatching. RESULTS: Murine blastocysts were comparatively biopsied using one of four methods and contrasted to zona slit and nonmicromanipulated controls. Operative cellular injury was assessed by uptake of fluorescein isothiocyanate (FITC). Postoperative embryonic viability was assessed by blastocoele reexpansion and hatching inner cell mass development and trophectoderm plating. All techniques yielded cells available for genetic analysis. CONCLUSIONS: The slit/excision technique and hatch/excision techniques exhibited lower operative injury and the higher postoperative viability than aspiration or incision. The slit/excision and the hatch/excision techniques, though requiring two operative steps, appear to be the least damaging of the four methods. Therefore, they should be most applicable to human blastocysts obtained either by extended culture in vitro or by uterine lavage.

Animals↗
PubMed1989

Modified photometric method for the determination of phospholipase A activities.

A modified photometric method for the determination of phospholipase A activities which is based on a previously published reaction principle is described. The modified assay uses a lyophilized substrate emulsion rather than a freshly prepared phospholipid emulsion and a new chromogen (tribromohydroxybenzoic acid and 4-amino-antipyrine) with a high molar absorption coefficient (1,930 m2/mol at 546 nm, 2,900 m2/mol at 512 nm wavelength). The new test is more practicable with respect to pipetting volumes and incubation times. Preliminary results of a method evaluation indicate that the modified assay fulfills the usual criteria for clinical chemical enzyme measurements.

Humans↗
PubMed1991

Pertussis toxin stimulation of catecholamine release from adrenal medullary chromaffin cells: mechanism may be by direct activation of L-type and G-type calcium channels.

We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca2+]o-dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca(2+)-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca(2+)-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K+]o, nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba2+]o. All these data are consistent with the concept that PTX may act on Ca2+ channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca2+ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca2+ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca2+ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca2+ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca(2+)-channel conductance (6 nS/cell at 2.6 mM [Ca2+]o). PTX evoked the activation of a new class of Ca(2+)-selective channels (5 pS in 25 mM [Ca2+]pipet), which are rather insensitive to membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

A practicable variant of the ion exchange method for the radiometric estimation of ornithine decarboxylase activity.

A known ornithine decarboxylase assay working with ion exchange separation of [3H]ornithine and [3H]putrescine has been revised. The assay can be performed in disposable 1.5 ml vessels with a total of four pipetting steps. The separation of enzyme substrate and product, respectively, requires 3 h per 50 samples. The detection limit is about 50 pmoles [3H]putrescine formed.

Animals↗
PubMed1993

A homogeneous immunological detection system for soman using the in vitro protection of acetylcholinesterase by a monoclonal antibody.

In this study a monoclonal antibody (MAb) based soman detection system was investigated. Since the MAb F71D7 recognizes the pinacolyl group of soman, non-toxic soman analogues are also detected when using an indirect competitive ELISA. This can lead to falsely positive results. The toxic effect of soman is, however, independent of the pinacolyl group. In the described homogeneous enzyme immunoassay (EIA), the inhibitory effect of soman on acetylcholinesterase (AChE) was combined with its specific binding to the MAb F71D7 in order to minimize false positive results and enhance the specificity of the detection system. In this rapid EIA no incubation or washing steps are necessary, so only time for pipetting and reaction have to be considered. Soman could be detected in concentrations of 1.6-25 nM using the EIA. This corresponds to 8 pg soman per 25 microliters sample and means that compared to other ELISA systems, besides enhanced specificity, the limit of detection could be improved by 3 orders of magnitude.

Acetylcholinesterase↗
PubMed1994

Cryopreservation of blastomeres separated from two-cell mouse embryos by an ultrarapid freezing method.

PURPOSE: To clarify the developmental capacity of frozen two-cell blastomeres, we investigated in vivo and in vitro viabilities of blastomeres that were frozen ultrarapidly after separation from two-cell mouse embryos. Two-cell embryos obtained from superovulated F1 hybrid females were denuded by treatment with 0.5% pronase solution and then induced to separate into two single blastomeres by gentle pipetting. The blastomeres were cryopreserved by an ultrarapid freezing method. RESULTS: The preimplantation developmental rate of two-cell embryos frozen in 3.0 M DMSO was significantly higher than the rate of those frozen in 15 and 4.5 M DMSO (at least P < 0.05). The in vitro developmental rate of the ultrarapidly frozen-thawed blastomeres separated from two-cell embryos (75.0%) was similar to that of nonfrozen blastomeres (76.0%). When eight pairs of blastocysts that developed from frozen two-cell mouse blastomeres were transferred to pregnant ICR recipients on Day 3, four live singletons were born. CONCLUSION: Thus, the results indicate that two-cell mouse blastomeres can be frozen by the ultrarapid freezing method.

Animals↗
PubMed1994

Electron microscopic study of human sperm membrane isolation.

OBJECT: Our purpose was to isolated pure, homogeneous human sperm membranes, free of cellular contaminants. METHODS: Donor semen samples collected after masturbation were stored at -70 degrees C and eventually pooled. Each attempt at sperm membrane isolation required 800 x 10(6) spermatozoa which were sonicated by ultrasound (40% output; Vibra Cell). The effect of sonication time (3 x 5, 3 x 15, and 180 sec) on membrane isolation was investigated. Sonicated samples were centrifuged (500 g, 5 min) and the supernatant was pipetted off. The supernatant of the centrifuged sample was layered on either a sucrose cushion (supernatant on 1.6 M sucrose) or a discontinuous sucrose gradient and centrifuged (100,000 g, 1 hr). Contents of supernatants of sonicated samples and fractions (sucrose interfaces) were then fixed in 1.0% tannic acid and 2.5% buffered glutaraldehyde and examined electron microscopically using standard procedures. RESULTS: (1) The optimal sonification time was found to be 3 x 15 sec. (2) Membrane isolation using a sucrose cushion was found to be inadequate, showing significant cellular contamination. (3) Sperm membrane isolation from the sucrose interface between 0.75 and 1.05 M sucrose was found to be most effective. CONCLUSION: The advantage of this method is its simplicity. The drawback of this method is the large number of spermatozoa required for membrane purification.

Cell Fractionation↗
PubMed1989

Culture of human corpus cavernosum endothelium.

A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissues whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin- or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344-354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3 x 0.3 x 0.5 cm) usually produces 120 cm2 of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7 x 10(4) cells/cm2. The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum.

Cell Division↗
PubMed1983

Pepsin can be used to subculture viable mammary epithelial cells.

Normal mouse mammary epithelial cells in primary culture can be passaged as viable single cells using 0.5 to 1.0 mg/ml pepsin in Hanks' salt solution. After 5 min the pepsin treatment preferentially removes fibroblasts, leaving a monolayer of purified epithelial cells that can be removed by pipetting and transferred to new culture vessels or injected into animals.

Animals↗
PubMed1996

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF.

To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

Animals↗
PubMed1999

Quantifying the geometry of micropipets.

Accurate knowledge of the internal diameter (id) of micropipet tips is important, because the ability to study many different aspects of biological membranes is a very sensitive function of tip size. The authors examined two methods used to characterize pipet tips: the digital manometric method (DMM) and bubble number method (BNM). For DMM, the authors compared the ability of Laplace's equation (model I) and a modified form of his equation (model II), which accounts for adhesion between the test fluid and glass. Pressure measurements were made with a digital manometer, and ids at the tip were measured using scanning electron microscopy (SEM). The micropipet tips showed a slight asymmetry in id, with a approx 5% difference between maximum and minimum id. On average, model I overestimates the largest id by 2%. Model II overestimates the smaller id by 2%. For micropipet tips ranging from 1.00 to 5.00 microm, the corresponding uncertainties range from 20 to 100 nm. Making the normally hydrophilic glass surface hydrophobic strongly reduced threshold pressures when tested in water, but not 100% methanol. Compared to BNM, DMM was insensitive to changes in atmospheric pressure: BNM can be corrected for changes in atmospheric pressure. Convergence angle(s) can be determined from measurements of the pressure and the axial distance of the meniscus from the tip. The accuracy and precision of digital manometry approaches that of SEM. DMM should be particularly useful in selecting micropipets for patch clamp studies of small vesicles (< 10 microm), and may enable systematic selection of micropipets for many other experiments.

Atmospheric Pressure↗
PubMed1976

Spread and control of mycoplasmal infection of cell cultures.

Environmental sampling was performed during trypsinization and passage of 3T-6 cell cultures that contained a mean of 4.3 X 10(7) colony forming units (CFU) per ml supernatant of A. laidlawii. The lip of the culture flask and the outside of the used pipet were always heavily contaminated. The outside of the culture flask (3/7), the work surface (8/12) and the outside of a pan of disinfectant (4/5) were regularly contaminated with mycoplasmas. Airborne mycoplasmas were detected eight of 32 times (25%) by settling plates; simultaneous forced-air samplers by two different methods were always negative. The technician's hands were contaminated two of 15 samples. When hands were contaminated, more contamination was detected in the environment. Droplets of A. laidlawii and M. orale inoculated onto work surfaces survived drying for a minimum of 3 days, even in laminar airflow cabinets. Twenty-five of 31 (80.6%) cell culture technicians carried M. salivarium in their throats; only two carried M. orale. It is concluded that mycoplasma-infected clltures are the most common source of further infection. Recommendations for prevention and control of mycoplasmal infection are listed.

Acholeplasma laidlawii↗
PubMed1990

Localization of calcium and microfilament changes in mechanically stressed cells.

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.

Actin Cytoskeleton↗
PubMed2005

Determination of Iron in Caco-2 cells by ET-AAS.

An electrothermal atomic absorption spectrometric method (ET-AAS) was developed for the direct determination of iron in intestinal Caco-2 cells after studying cell viability and proliferation using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT test). Zeeman background correction and end-capped graphite tubes with L'vov platforms were used. Samples were dissolved in dimethylsulfoxide (DMSO) and pipetted directly into the graphite tube. The preashing, pretreatment and atomization steps were optimized. The temperatures selected were 600, 1200, and 2100 degrees C, respectively. Stability measurements were performed using iron standard solutions in DMSO on the one hand and acidified cell solutions on the other. Direct measurement and standard addition were compared in order to determine possible influences of the matrix. The low detection limit of the ET-AAS method (1.3 microg/L or 3.3 microg/g) combined with the small sample quantities required are ideal for the determination of iron in cells due to the low iron content and the limited growth area of the cells. The method was developed for iron uptake studies for toxicological purposes.

Caco-2 Cells↗
PubMed2005

A direct solid sampling electrothermal atomic absorption spectrometric method for determination of trace elements in zirconium dioxide.

A direct solid sampling electrothermal atomic absorption spectrometric method for determination of Cd, Co, Cr, Cu, Fe, Li, Mn, Ni and Zn in zirconium dioxide has been developed. Using optimised experimental conditions, very effective in-situ analyte/matrix separation was achieved without any chemical modification. After the measurement, the matrix residue could easily be tipped out from the platform. In the determination of Cr, before sampling, the platform bottom was covered with carbon powder. Quantification was performed using calibration curves measured with aqueous standard solutions pipetted onto the matrix residue from a previous sample run. Sample amounts between 0.5 and 40 mg were applied for each analysis cycle. The accuracy was determined by comparison of the results with those obtained by radiochemical neutron activation analysis and by electrothermal atomic absorption spectrometry using liquid sampling of digests and slurry sampling. For the nine elements assayed, the limits of detection achievable by this method are between 0.06 ng g(-1) (Cd) and 2.3 ng g(-1) (Fe).

Metals↗
PubMed2000

ETAAS method for the determination of Cd, Cr, Cu, Mn and Se in blood fractions and whole blood.

An electrothermal atomic absorption method (ETAAS) for the direct determination of trace elements (Cd, Cr, Cu, Mn, Se) both in blood fractions (erythrocytes, plasma and lymphocytes) and whole blood was developed. Zeeman background correction and graphite tubes with L'vov platforms were used. Samples were diluted with HNO3/Triton X-100 and pipetted directly into the graphite tube. Ashing, pretreatment and atomization steps were optimized carefully for the different fractions and elements applying different matrix modifiers for each element. For the lymphocyte fraction a multi-fold injection technique was applied. Low detection limits of the ETAAS method (Cd 0.13 microgram/L, Cr 0.11 microgram/L, Cu 0.52 microgram/L, Mn 0.13 microgram/L, Se 0.7 microgram/L of whole blood) combined with small quantities of sample necessary for analysis allow determination of trace elements in this matrix. Verification of possible differences in the trace element status of humans was performed with statistical significance (P < 0.05). In addition, a contribution to the determination of normal values of essential elements was achieved. The method was applied for determination of trace elements in human blood and blood fractions of two groups (n = 50) different in health status.

Erythrocytes↗
PubMed1998

Design, construction and six years' experience of an integrated system for automated handling of discrete blood samples.

The present paper describes the design of an integrated system to aid in the taking and measurement of manual blood samples during nuclear medical examinations requiring blood sampling. In contrast to previously published systems, the present system is not used in the actual sampling of the blood, but aims to aid in all other aspects of handling and measurement. It consists of two main parts. One part is a distributed software system running on the scanner host computer used to register sample times, to display information pertaining to the ongoing examination and to collect data from a number of well crystals. The other main part consists of an industrial robot used to perform the actual weighing, centrifugation, pipetting and measurement of the samples. The system has been operational for 6 years, during which time it has had an "up-time" in excess of 95% and has handled and measured the blood samples from more than 5000 examinations, each comprising an average of 15 blood samples. The throughput of the system is 50 whole blood samples or 21 plasma samples per hour. In addition it has to a large extent removed the "human factor" from the process, thereby increasing the reliability of the data.

Automation↗
PubMed2004

Isolation and some properties of fimbriae of oral Streptococcus intermedius.

Streptococcus intermedius 1208-1 carried linear fiber-like fimbriae that extended radially from the cell surface. The fimbriae were isolated by pipetting and sonication and were purified by ammonium sulfate precipitation followed by a column chromatography series. Heat treatment in the presence of sodium dodecyl sulfate resulted in the dissociation into smaller molecules. Rabbit antiserum raised against the purified protein reacted with fimbriae on the surface of bacteria under immunogold staining. Serotype g or g-related strains produced the fimbriae and aggregated in human saliva. The aggregation was inhibited by the anti-fimbriae immunoglobulin Fab fragment or the purified fimbriae.

Antibodies, Bacterial↗