assay Dotaz Zobrazit nápovědu
- Klíčová slova
- BIOLOGICAL ASSAY *,
- MeSH
- biotest * MeSH
- Publikační typ
- časopisecké články MeSH
Fluorometric glutathione assays have been generally preferred for their high specificity and sensitivity. An additional advantage offered by fluorescent bimane dyes is their ability to penetrate inside the cell. Their ability to react with glutathione within intact cells is frequently useful in flow cytometry and microscopy. Hence, the aims of our study were to use monochlorobimane for optimizing a spectrofluorometric glutathione assay in cells and then to compare that assay with the frequently used ortho-phthalaldehyde assay. We used glutathione-depleting agents (e.g., cisplatin and diethylmalonate) to induce cell impairment. For glutathione assessment, monochlorobimane (40μM) was added to cells and fluorescence was detected at 394/490nm. In addition to the regularly used calculation of glutathione levels from fluorescence change after 60min, we used an optimized calculation from the linear part of the fluorescence curve after 10min of measurement. We found that 10min treatment of cells with monochlorobimane is sufficient for evaluating cellular glutathione concentration and provides results entirely comparable with those from the standard ortho-phthalaldehyde assay. In contrast, the results obtained by the standardly used evaluation after 60min of monochlorobimane treatment provided higher glutathione values. We conclude that measuring glutathione using monochlorobimane with the here-described optimized evaluation of fluorescence signal could be a simple and useful method for routine and rapid assessment of glutathione within intact cells in large numbers of samples.
- Klíčová slova
- Cell impairment, Fluorescence, Glutathione assay, Monochlorobimane, Ortho-phthalaldehyde,
- MeSH
- biotest ekonomika metody MeSH
- buněčné linie MeSH
- cisplatina toxicita MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie ekonomika metody MeSH
- glutathion analýza metabolismus MeSH
- lidé MeSH
- malonáty toxicita MeSH
- o-ftalaldehyd chemie MeSH
- průtoková cytometrie MeSH
- pyrazoly chemie MeSH
- senzitivita a specificita MeSH
- studie proveditelnosti MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cisplatina MeSH
- diethyl malonate MeSH Prohlížeč
- fluorescenční barviva MeSH
- glutathion MeSH
- malonáty MeSH
- monochlorobimane MeSH Prohlížeč
- o-ftalaldehyd MeSH
- pyrazoly MeSH
The rapid accumulation of sequence data and powerful protein engineering techniques providing large mutant libraries have greatly heightened interest in efficient methods for biochemical characterization of proteins. Herein is reported a continuous assay for screening of enzymatic activity. The assay is developed and tested with the model enzymes haloalkane dehalogenases and relies upon a fluorescent change of a derivative of 8-hydroxypyrene-1,3,6-trisulphonic acid due to the pH drop associated with the dehalogenation reactions. The assay is performed in a microplate format using a purified enzyme, cell-free extract or intact cells, making the analysis quick and simple. The method exhibits high sensitivity with a limit of detection of 0.06 mM. The assay is successfully validated with gas chromatography and then applied for screening of 12 haloalkane dehalogenases with the environmental pollutant bis(2-chloroethyl) ether and chemical warfare agent sulfur mustard. Six enzymes exhibited detectable activity with both substrates. The within-day variability of the assay for five replicates (n = 5) was 21%.
- Klíčová slova
- activity screening, enzyme assay, fluorescence, haloalkane dehalogenase, sulfur mustard,
- MeSH
- biotest metody MeSH
- fluorescenční barviva chemie MeSH
- hydrolasy chemie MeSH
- koncentrace vodíkových iontů MeSH
- látky znečišťující životní prostředí chemie MeSH
- proteinové inženýrství metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fluorescenční barviva MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
- látky znečišťující životní prostředí MeSH
Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.
- Klíčová slova
- Calibration, Comet assay, Radiation, Ring-trial, Standardisation,
- MeSH
- ionizující záření * MeSH
- kalibrace MeSH
- kometový test metody MeSH
- lidé MeSH
- poškození DNA * MeSH
- savci MeSH
- vztah dávky záření a odpovědi MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between 'desirable' and 'essential' information: 'essential' information refers to the precise details that are necessary to assess the quality of the experimental work, whereas 'desirable' information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.
- MeSH
- dodržování směrnic statistika a číselné údaje MeSH
- kometový test metody normy MeSH
- konsensus MeSH
- laboratoře MeSH
- lidé MeSH
- výzkumný projekt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Four laboratories, two in the Czech Republic (Brno and Prague) and two in the CIS (Moscow and Duschanbe), participated in the International Programme on Chemical Safety's (IPCS) collaborative study to evaluate the utility of the most commonly used plant test systems, including the Arabidopsis thaliana assay, for assessing the mutagenic potential of environmental agents. Out of the five compounds evaluated in the Arabidopsis assay, three compounds, i.e., ethyl methanesulfonate, N-methyl-N-nitrosourea, and azidoglycerol, were reported to be mutagenic by all four participating laboratories. Sodium azide (NaN3) demonstrated a negative response in all four laboratories, whereas maleic hydrazide was reported to be weakly mutagenic by one laboratory and nonmutagenic by the other three laboratories.
- MeSH
- Arabidopsis embryologie genetika MeSH
- azid sodný MeSH
- azidy toxicita MeSH
- biotest metody MeSH
- ethylmethansulfonát toxicita MeSH
- hydrazid kyseliny maleinové toxicita MeSH
- methylnitrosomočovina toxicita MeSH
- mezinárodní spolupráce MeSH
- monitorování životního prostředí metody MeSH
- mutageny analýza toxicita MeSH
- propylenglykoly toxicita MeSH
- reprodukovatelnost výsledků MeSH
- testy genotoxicity metody MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- Názvy látek
- azid sodný MeSH
- azidoglycerol MeSH Prohlížeč
- azidy MeSH
- ethylmethansulfonát MeSH
- hydrazid kyseliny maleinové MeSH
- methylnitrosomočovina MeSH
- mutageny MeSH
- propylenglykoly MeSH
Ellman's assay is the most commonly used method to measure cholinesterase activity. It is cheap, fast, and reliable, but it has limitations when used for biological samples. The problems arise from 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), which is unstable, interacts with free sulfhydryl groups in the sample, and may affect cholinesterase activity. We report that DTNB is more stable in 0.09 M Hepes with 0.05 M sodium phosphate buffer than in 0.1M sodium phosphate buffer, thereby notably reducing background. Using enzyme-linked immunosorbent assay (ELISA) to enrich tissue homogenates for cholinesterase while depleting the sample of sulfhydryl groups eliminates unwanted interactions with DTNB, making it possible to measure low cholinesterase activity in biological samples. To eliminate possible interference of DTNB with enzyme hydrolysis, we introduce a modification of the standard Ellman's assay. First, thioesters are hydrolyzed by cholinesterase to produce thiocholine in the absence of DTNB. Then, the reaction is stopped by a cholinesterase inhibitor and the produced thiocholine is revealed by DTNB and quantified at 412 nm. Indeed, this modification of Ellman's method increases butyrylcholinesterase activity by 20 to 25%. Moreover, high stability of thiocholine enables separation of the two reactions of the Ellman's method into two successive steps that may be convenient for some applications.
- Klíčová slova
- Acetylcholinesterase, Biological samples, Butyrylcholinesterase, DTNB, Ellman’s assay,
- MeSH
- butyrylcholinesterasa metabolismus MeSH
- ELISA metody MeSH
- enzymatické testy metody MeSH
- kyselina dithionitrobenzoová metabolismus MeSH
- lidé MeSH
- thiocholin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- butyrylcholinesterasa MeSH
- kyselina dithionitrobenzoová MeSH
- thiocholin MeSH
Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.
- Klíčová slova
- BIOLOGICAL ASSAY *, ESCHERICHIA COLI *, EXPERIMENTAL LAB STUDY *, LYSINE *,
- MeSH
- biotest * MeSH
- Escherichia coli * MeSH
- lysin * MeSH
- výzkum * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- lysin * MeSH
A method to cultivate anaerobic bacteria on standard 96-well microplates with automatic recording of growth curves is presented. The method was used as a kinetic growth inhibition assay with denitrifying bacteria Paracoccus denitrificans, and applied to various heavy metal ions and selected agrochemicals. Incorporated in a battery of other biotest the assay could take into account effects of toxicants on denitrifying organisms. The results (EC50) revealed that the assay was relatively sensitive. Performed in vials, the assay was also applied to toxicity testing of volatile compounds and represented a convenient method for assessing samples containing volatile constituents.
- MeSH
- agrochemikálie toxicita MeSH
- biotest MeSH
- dusík metabolismus MeSH
- kinetika MeSH
- kultivační média MeSH
- Paracoccus účinky léků růst a vývoj metabolismus MeSH
- pesticidy toxicita MeSH
- Pseudomonas účinky léků růst a vývoj metabolismus MeSH
- těžké kovy toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- agrochemikálie MeSH
- dusík MeSH
- kultivační média MeSH
- pesticidy MeSH
- těžké kovy MeSH
