TbTryS (1.44 µM, 2.4 × 10−5 µmol/min.mL) and 10 µM Ebselen (stock prepared at 100 µM in water with DMSO 20% v/v) were mixed and incubated during 1 h at 20–23 °C. Thereafter, the excess compound was removed with a Zeba Spin Desalting Column 7 kDa Molecular Weight Cut-Off (Thermo Fisher Scientific) previously equilibrated with reaction buffer. Next, samples (10 µL) were incubated for 30 min in the absence or presence of DTT 5 mM for 30 min at 28 °C. TryS activity was measured in a 384-plate upon diluting the samples in reaction buffer (1:2 dilution) and incubating for 1 h at 28 °C with MM added or not of DTT 5 mM (stock prepared at 80 mM in reaction buffer), respectively. Controls with and without enzyme along with DMSO at 2% (v/v) were included. The reaction was stopped with the addition of the BIOMOL GREENTM reagent. The absorbance was measured at 620 nm with a MultiScan FC plate reader (Thermo Fisher Scientific). Six replicates were tested per sample and the % TryS inhibition calculated as indicated in the section High-throughput TryS assay .
Multiscan fc
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland, Germany, Japan, China
The Multiscan FC is a microplate reader designed for a wide range of absorbance-based assays. It features a UV/Vis wavelength range and can accommodate various microplate formats. The Multiscan FC provides accurate and reliable data for your laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8, by Beyotime (5 mentions)
High binding clear polystyrene 96 well plates, by Thermo Fisher Scientific (4 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (4 mentions)
Skanit for multiscan fc, by Thermo Fisher Scientific (3 mentions)
Edu imaging kit, by Thermo Fisher Scientific (3 mentions)
Xtt assay, by Roche (2 mentions)
Skanit for multiscan fc software, by Thermo Fisher Scientific (2 mentions)
Dmi3000b microscope, by Leica (2 mentions)
96 well plates, by Euroclone (2 mentions)
Mtt reagent, by Merck Group (2 mentions)
Cck 8 kit, by Beyotime (2 mentions)
Tween 20, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse fc antibody, by Abcam (2 mentions)
Mts reagent, by Promega (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Nih3t3 cells, by American Type Culture Collection (1 mentions)
Cell counting kit 8 cck 8 reagent, by Dojindo Laboratories (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Streptavidin horseradish peroxidase conjugate, by GE Healthcare (1 mentions)
158 protocols using multiscan fc
1
Enzymatic Inhibition Assay for Trypanothione Synthase
2
Colorimetric GlmU Acetyltransferase Inhibition Assay
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A colorimetric assay coupled with 5,5′-dithio-bis-(2-nitrobenzoic acid [DTNB]) was performed to screen inhibitors of GlmU acetyltransferase.26 (link) According to the method in our previous work, the enzymatic reaction was a 50 μL mixture performed in a 96-well microtiter plate containing 50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 0.4 mM glucosamine-i-phosphate (GlcN-1-P), 0.4 mM acetyl CoA (Ac-CoA) and purified GlmU protein at 37 °C for 5 min. The reaction was then terminated by adding 50 μL 6 M guanidine hydrochloride and 50 μL Ellman's reagent (0.2 mM DTNB and 1 mM ethylenediaminetetraacetic acid [EDTA] in 50 mM Tris–HCl, pH 7.5). The absorbance value was measured at a wavelength of 405 nm by using Multiscan Fc (Thermo scientific). Separately, the “background group” containing the substrates (Ac-CoA and GlcN-1-P) and tested compound was used to correct the error. Percent inhibition: inhibition (%) = 100 (1 − [A405 − AMin]/[AMax − AMin]) for which A405 was the absorbance of the test reaction that contained compound, AMin was the absorbance of the background group, and AMax was the absorbance of the uninhibited reaction. GraphPad was used to calculate the concentration of 50% inhibition (IC50).
3
Cell Viability Assays for Neuroblastoma and Melanoma
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Cell viability was determined by XTT assay (Roche Diagnostic, USA), which measures the cells’ ability to reduce the tetrazolium salt XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) to a water-soluble formazan product. Cells were seeded at a density of 5 × 103 neuroblastoma or Ab melanoma and 50 × 103 Ma melanoma cells into 96-well plates with the suitable cultivation media. After 24 h, the media were exchanged and cells were stimulated with appropriate concentrations (1; 10; 20; 40; 50; 100; 150 µM) of ART for 48 and 72 h. The orange-colored formazan product was quantified at 450 nm in a microplate reader (Multiscan FC, Thermoscientific USA). Cell viability was normalized with respect to an untreated control (100%).
In the Trypan Blue test, the cell suspension was mixed with an equal volume of 0.4% isotonic Trypan Blue solution and the total number of cells and fraction of nonviable, dye-accumulating cells was counted after 2 min in a Fuchs–Rosenthal hemocytometer under a light microscope.
In the Trypan Blue test, the cell suspension was mixed with an equal volume of 0.4% isotonic Trypan Blue solution and the total number of cells and fraction of nonviable, dye-accumulating cells was counted after 2 min in a Fuchs–Rosenthal hemocytometer under a light microscope.
4
Bacterial Growth Conditions Optimization
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Growth densities to each strain were measured in temperature of +37°C and room temperature with 230 rpm shaking or without shaking. Cells were grown overnight in 5 ml of LB media (+37°C, 230 rpm) and then transferred into 5 ml of fresh LB media in 1:5,000 ratio. Fresh cultures were grown in experimental settings of +37°C and 230 rpm or RT and 0 rpm for 20 h and growth densities were calculated as colony forming units (cfu)/ml. As a standard initial liquid culture for the growth curve experiments all the strains were cultured in 5 ml of 100% LB, +37°C, and 230 rpm overnight and then transferred into experimental settings. In order to test the effect of shaking, the initiating cultures were prepared then transferred into 5 ml of LB in 1:100 ratio and grown in experimental settings of +37°C and 230 rpm and +37°C and 0 rpm. In growth curve experiments the effect of different media and varying concentrations and compositions of nutrients on growth was determined for each strain. One hundred percent LB was used throughout the experiments unless mentioned otherwise. Growth curves were measured in 10 and 1% LB, 100% BHI, and 100% of pure DMEM by diluting the initial culture in 1:100. Growth curves were measured at +37°C, 595 nm wavelength with Multiscan FC (Thermo Scientific) for 20 h in 5 min intervals and maximum growth and average growth rate were calculated.
5
Quantifying IL-17A and TGF-β by ELISA
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IL-17A and TGF-β levels were measured by ELISA according to the manufacturer’s standard protocols (eBioscience, San Diego, CA). Absorbance was read on a Multiscan FC plate reader and analyzed with SkanIt for Multiscan FC software (Thermo Scientific, Schwerte, Germany).
6
Cytotoxicity and Proliferation Assay of ISL on HBFs
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For the determination of the cytotoxic/cytostatic effect of ISL on HBFs we seeded cells at a concentration of 2.5 × 104 cells/cm2 in culture medium into microplates (96 wells, flat bottom; VWR). 24 h after seeding, we added different concentrations of ISL (12.5 μM, 25 μM, 50 μM, 75 μM, 100 μM). After the incubation period (24 h, 72 h and 4 days), we performed the MTT assay (due to manufacturer protocol) for determination of cytotoxicity and Crystal Violet assay for determination of cells’ proliferation. The absorbance of the samples was measured using a microplate reader (Multiscan FC; Thermo Fisher Scientific). The wavelength to measure absorbance of the formazan product was 570 nm and for crystal violet was 540 nm. IC50 was calculated for each time of incubation with ISL.
7
Cytotoxicity Evaluation of PEG-NaGdF4:Yb,Er NPs
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The cytotoxicity of the as-prepared PEG-NaGdF4:Yb3+,Er3+ NPs was evaluated in vitro through an MTT assay using NIH3T3 cells (ATCC). The NIH3T3 cells were seeded at a density of 104 cells/well in a 96-well plate and cultured in a minimum essential medium containing 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a humidified atmosphere with 5% CO2. The cells were exposed to different concentrations of the PEG-NaGdF4:Yb3+,Er3+ NPs, ranging from 0.01 to 10 mM, for 24 h at 37°C. The cells were then washed with 9.6 mM PBS. A mixture of water-soluble tetrazolium salts, WST-8, and the culture medium was added to each well, and the cells were incubated for 30 min. The cell viability was evaluated by measuring the absorption of WST-8 at 450 nm in a microplate reader (Multiscan FC, Thermo Scientific, Massachusetts, US). The experiment was repeated five times.
8
Cytokine Quantification by ELISA
9
Cytokine and Angiogenic Factor Quantification
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Tumor necrosis factor-alpha (TNF-a), interleukin (IL)-1β, IL-6, PRDX2 and VEGF secretion in cell culture supernatants or serum were measured by ELISA, according to the manufacturer’s standard protocols (eBioscience, San Diego, CA). Absorbance was read on a Multiscan FC plate reader and analyzed with Skan It for Multi scan FC software (Thermo Scientific, Schwerte, Germany).
10
Cell Proliferation Assay in U2OS and MG63 Cells
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U2OS and MG63 cells were first seeded into 96‐well tissue culture plates (3 × 103 cells per well). The detection was performed at 0, 24, 48 and 72 hours. After adding 10 µL of Cell Counting Kit‐8 (CCK8) reagent (Dojindo Molecular Technologies, Inc, Kumamoto, Japan) per well and incubating for 2 hours, the absorbance at 450 nm was measured using a Multiscan FC plate reader and analysed with SkanIt for Multiscan FC software (Thermo Scientific).
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