MDA levels were determined in plasma as described previously [32 (link)]. After heating (60 min, 100 °C), plasma samples were neutralized with methanol/NaOH, centrifuged (3 min, 3000 rpm) and their MDA was measured with HPLC (excitation: λ 532 nm, emission: λ 563 nm, LaChrom Merck Hitachi Chromatography System, Vienna, Austria; HPLC column 125 × 4 mm, 5 μm; Merck, Vienna, Austria). The antioxidant capacity of serum was measured via the ferric reducing ability potential (FRAP) assay as described earlier [24 (link)] in triplicates, using trolox as a standard. Absorbance was measured with BMG FLUOstar OPTIMA Microplate Reader (BMG LABTECH GmbH) at 593 nm and results are expressed as trolox equivalents in μmol/L. GSSG (glutathione disulfide) and GSH were analyzed with use of N-Ethylmaleimide and O-phthalaldehyde according to an adopted method of Hissin and Hilf [33 (link)] as described previously [34 (link)]. All samples were analyzed fluorometrically in triplicates with external standards of GSSG and GSH using BMG FLUOstar OPTIMA Microplate Reader (BMG LABTECH GmbH).
Bmg fluostar optima microplate reader
Manufactured by BMG Labtech
Sourced in Germany, United States, Australia
The BMG FLUOstar OPTIMA Microplate Reader is a versatile and reliable instrument designed for performing various fluorescence, luminescence, and absorbance measurements in microplates. It offers a range of detection modes to accommodate a wide variety of assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase reporter assay kit, by Promega (3 mentions)
Substrate reagent pack, by R&D Systems (2 mentions)
Optima software 2, by BMG Labtech (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
E1500, by Promega (2 mentions)
Jetpei reagent, by Polyplus Transfection (1 mentions)
Celltiter blue ctb, by Promega (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Cytoselect 96 well cell transformation assay kit, by Cell Biolabs (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Amaxa nucleofector kit 5, by Lonza (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Porcine cholecystokinin elisa kit, by MyBioSource (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Amplite universal fluorimetric mmp activity assay kit, by AAT Bioquest (1 mentions)
Celltiter glo cell viability assay, by Promega (1 mentions)
Powerwave xs microplate spectrophotometer, by Agilent Technologies (1 mentions)
Any kd mini protean tgx stain free protein gel, by Bio-Rad (1 mentions)
27 protocols using bmg fluostar optima microplate reader
1
Quantification of Oxidative Stress Markers
2
Quantification of Oxidized and Reduced Glutathione
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Oxidized and reduced glutathione were measured using N-Ethylmaleimide and O-phthalaldehyde, as described previously41 (link). All samples were analysed in triplicates using external GSSG and GSH standards, in a 96-well plate fluorometer (BMG FLUOstar OPTIMA Microplate Reader, BMG LABTECH GmbH).
3
Amyloid-beta Seeding Assay for Brain Samples
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For these assays, a previously described protocol60 (link) - not yet fully validated – was used. It was slightly modified in order to comply with the analysis of brain samples.
5 microliters of brain homogenates’ S1 fractions were used as seeds and diluted in 100 mM Tris-HCl pH 7.5, 5 μM ThT, 4 μM synthetic Aβ1-42WT or Aβ1-42A2V. The reactions were transferred in quadruplicate into wells of a black, clear bottom, 96-well microplate (Nunc). The plate was incubated at 37 °C into a BMG Fluostar Optima Microplate Reader (BMG Labtech), and shaken every other minute. Fluorescence was measured every 15 minutes. The same protocol was used to test the seeding ability of brain extracts on Aβ1-40WT, except for Aβ1-40 concentration (10 μM) and incubation temperature (30°).
5 microliters of brain homogenates’ S1 fractions were used as seeds and diluted in 100 mM Tris-HCl pH 7.5, 5 μM ThT, 4 μM synthetic Aβ1-42WT or Aβ1-42A2V. The reactions were transferred in quadruplicate into wells of a black, clear bottom, 96-well microplate (Nunc). The plate was incubated at 37 °C into a BMG Fluostar Optima Microplate Reader (BMG Labtech), and shaken every other minute. Fluorescence was measured every 15 minutes. The same protocol was used to test the seeding ability of brain extracts on Aβ1-40WT, except for Aβ1-40 concentration (10 μM) and incubation temperature (30°).
4
Transfection and Luciferase Assay Protocol
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Transfections were performed using previously published methods44 (link). Cultures were grown to subconfluence in antibiotic-free media (10% FCS). Each transfection contained a pGL3-MMP promoter construct (1 μg), 1.5 μl jetPEITM reagent (Polyplus Transfection, Illkirch, France), and 10 ng phRL-SV40, encoding the internal standard Renilla luciferase. Following transfection, cells were treated with: IL-6, soluble IL-6Rα (Peprotech), PMA and/or human IFN-γ. Luciferase activity was recorded using the BMG FLUOstarOPTIMA microplate reader (BMG Labtech, Melbourne, Australia) using Renilla luciferase as a normalization standard.
5
Cytotoxicity Screening of Novel Compounds
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Stock solutions (32 mM) for compounds 1 , 2 , 3 , 4, and 5 were prepared using 100% DMSO as the solvent. Working solutions were then prepared at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µM by diluting stock solutions with culture medium. HEp2 cells were exposed to the working solutions up to 200 µM with 24 h incubation (5% CO2, 95% humidity, 37 °C). After 24 h treatment, the loading medium was removed, and cells were washed thrive with phosphate buffered saline (PBS) solution to remove residual compound. Then, medium containing 20% CellTiter Blue (CTB) (Promega, Madison, WI, USA) was added to the cells and allowed to incubate it for another 4 h. Following the incubation period, cell viability was assessed by determining fluorescence intensity at 570/615 nm using a BMG FLUOstar Optima microplate reader (BMG Labtech, Cary, NC, USA). The fluorescence intensity was normalized to 100% for untreated cells and the results are expressed as a percentage of viable cells.
6
Constructing Nanog-Luciferase Vectors
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To construct luciferase vectors containing Nanog promoter sequences, three different Nanog promoter sequences were amplified and inserted into a luciferase reporter plasmid before the Kozak and luciferase sequences (P1–P3: −2200–0, −1700–0, and −700–0 bp upstream of the Nanog coding sequence, respectively) using VectorBuilder services (VectorBuilder Inc., Chicago, IL, USA). The cJun-expressing and luciferase reporter plasmids were cotransfected into cells; empty vectors were used as controls. Luciferase signals were detected using a luciferase reporter assay kit (Promega, Madison, Wisconsin, USA) and measured using a BMG FLUOstar OPTIMA Microplate Reader (BMG LABTECH, Cary, North Carolina, USA).
7
Quantification of Cytokines and VEGF in RCC Cells
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IL6, IL8, IL11, IL15, and VEGF concentrations in cell culture supernatant of RCC cells and nonmalignant RC-124 cells were determined by use of target-specific DuoSet ELISA kits with a Substrate Reagent Pack containing stabilized hydrogen peroxide and tetramethylbenzidine (all R&D, Minneapolis, MN, USA) according to the supplier's instructions. Cells were sedimented (1.3 × g, 5 min), the supernatant was incubated with capture antibodies (overnight, 4°C), and soluble antigens were determined applying the BMG FLUOstar OPTIMA Microplate Reader with OPTIMA software 2.10 (BMG Labtech, Offenbach, Germany). Each sample was analyzed in duplicates.
8
Fluorescence-based Drug Quantification
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Firstly, a series of dilutions (at least six dilutions) of each model drug i.e. FS, FD 70, and FD 150 was prepared in PBS. The fluorescent emission of an aliquot of 150 μL sample of each drug solution was measured using a fluorescent plate reader (BMG FLUOstar OPTIMA Microplate Reader, BMG Labtech, Ortenberg, Germany) operated at 37 °C using 493 and 520 nm as excitation and emission wavelengths, respectively. For drug analysis, the gain was set at 1000 and 1500 for FS and FD 70/150, respectively. Standard calibration curves of fluorescence absorbance vs concentration (μg/ml) were constructed and used to calculate drug concentrations in the analyzed samples.
9
Cell Transformation Assay in Soft Agar
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Cell transformation and anchorage-independent growth assays were performed using a fluorescence-based CytoSelect 96-well cell transformation assay kit (Cell Biolabs, Inc., San Diego, CA, USA). KGN cells were plated in soft agar in a 96-well plate at 5000 cells/well and cultured for 20 days. COV434 cells were plated at 10 000 cells/well and incubated for 7 days. Cell transformation was determined according to the protocol provided by the manufacturer. Colony number was counted under a light microscope. The relative fluorescence unit (RFU) was determined with a BMG Fluostar Optima Microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).
10
Amyloid-beta Aggregation Kinetics Assay
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5 microliters of brain homogenates’ soluble fractions (S1) were diluted in 100 mM Tris-HCl pH 7.5, 5 μM ThT, and transferred in triplicate into wells of a black, clear bottom, 96-well microplate (Nunc). The plate was incubated at 25 °C into a BMG Fluostar Optima Microplate Reader (BMG Labtech). Every 59 minutes the plate was shaked for 1 minute and the fluorescence was measured. For the comparison of aggregation kinetics among Aβ extracted from different AD brains, we considered the slope of the curve described by fluorescence values at different time points.
A preliminary step in the ThT assays was used to test the effects of the initial quantity of soluble Aβ in AD brain extracts on the shape of aggregation curves. To this end, 5 or 10 microliters of brain homogenates’ soluble fractions (S1) of some human brain samples were analyzed by ThT assay and showed overlapping profiles.
A preliminary step in the ThT assays was used to test the effects of the initial quantity of soluble Aβ in AD brain extracts on the shape of aggregation curves. To this end, 5 or 10 microliters of brain homogenates’ soluble fractions (S1) of some human brain samples were analyzed by ThT assay and showed overlapping profiles.
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