H9c2 cardiomyoblasts and cardiomyocytes were seeded in 24-well plate. After 16 h, the medium was removed, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) solution (5 mg/mL in PBS) was added to each 24-well plate and incubated for 2 h at 37°C. Then, formazan salts were solubilized with DMSO and absorbance at 570 nm was measured using an ELISA plate reader (Multiskan EX; Thermo, Waltham, MA). The absorbance of each well was measured at 570 nm using an ELISA plate reader (Multiskan EX; Thermo, Waltham, MA).
Multiskan ex
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland, Germany, United Kingdom, Italy, China, France, India, Australia
The Multiskan EX is a microplate reader designed for absorbance-based measurements in life science applications. It can be used to measure absorbance in 96-well microplates, providing data on the optical density of samples. The Multiskan EX is capable of performing essential lab functions such as photometric and colorimetric assays.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (33 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (20 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Calcusyn, by Biosoft (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Cell proliferation reagent wst 1, by Roche (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (5 mentions)
Mtt assay, by Merck Group (5 mentions)
Wst 1, by Roche (4 mentions)
Griess reagent, by Merck Group (4 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (4 mentions)
Ascent software, by Thermo Fisher Scientific (4 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (4 mentions)
Prism 5, by GraphPad (4 mentions)
Ez cytox reagent, by Daeil Lab Service (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Dulbecco s phosphate buffered saline dpbs, by Lonza (3 mentions)
547 protocols using multiskan ex
1
MTT Assay for Cell Viability
2
Rat Hypocretin ELISA Assay
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Rat hypocretin ELISA kits were obtained from Wuhan Fine Biotech Co., Ltd. (Wuhan, China) and the procedures followed the standard instructions provided by the manufacturers. Absorbance was measured by an ELISA plate reader (Multiskan EX, Thermo Electron Corp., Waltham, MA, USA) with the wavelength set at 450 nm. The sensitivity is <4.688 pg mL−1, the assay range is between 7.8 and 500 pg mL−1, the intra-assay coefficient of variation is <8% and the inter-assay coefficient of variation is <10%.
3
BrdU-based Cellular Proliferation Assay
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The rate of cellular proliferation was estimated through incorporating pyrimidine analogue BrdU (5-bromo-2′-deoxyuridine), in place of thymidine, into the DNA of proliferating cells, using the Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche Applied Science, Mannheim, Germany). The antibody conjugated anti-BrdU-peroxidase binds incorporated BrdU. The complex BrdU/anti-BrdU-peroxidase was detected by the reaction between peroxidase conjugated to the BrdU antibody and the substrate (3,3',5,5'-tetramethylbenzidine). After reaching a satisfactory color intensity (after incubating between 5 and 30 min), the reaction was stopped with 1 M H2SO4 solution.
The cells were seeded in 96-well plates (TPP, Trasadingen, Switzerland) at a density of 1×104 cells per well and were maintained in 200 μL of previously described media formulations (NC1-NC4), supplemented by 10% FBS. After 48 h of incubation, the assay was performed according to the manufacturer's instructions. The reaction product (3,3',5,5'-tetramethyl-benzidine diimine) was quantified by measuring absorbance using a microplate reader Multiskan EX (Thermo Electron Corporation, Shanghai, China) set at 450 nm (reference wavelength: 620 nm). Cell proliferation was expressed as a percentage of the cells grown under condition NC1 (high glucose +l -glutamine medium).
The cells were seeded in 96-well plates (TPP, Trasadingen, Switzerland) at a density of 1×104 cells per well and were maintained in 200 μL of previously described media formulations (NC1-NC4), supplemented by 10% FBS. After 48 h of incubation, the assay was performed according to the manufacturer's instructions. The reaction product (3,3',5,5'-tetramethyl-benzidine diimine) was quantified by measuring absorbance using a microplate reader Multiskan EX (Thermo Electron Corporation, Shanghai, China) set at 450 nm (reference wavelength: 620 nm). Cell proliferation was expressed as a percentage of the cells grown under condition NC1 (high glucose +
4
Colorectal Cancer Cell IC50 Assay
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To calculate the half-maximal inhibitory concentration (IC50) values of the extract, cells were seeded in sterile 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark) at high density (1.5 × 104 cells/well) and incubated at 37 °C with 5% CO2 for 24 h to allow cell adhesion. Increasing concentrations of the extract (31.25–2000 μg/mL) and 5-fluorouracil (5-FU) as positive control (1.95–125 μg/mL) were added in the corresponding wells and were incubated for 48 h at 37 °C with 5% CO2. All the concentrations evaluated were performed in sextuplicate. The effect of the extract on tumor colorectal cell lines (HT-29, T-84 and SW-837) was evaluated using the Sulforhodamine-B (SRB) method [23 (link)]. Optical density values were determined by colorimetry at 490 nm using a microplate reader (Multiskan EX, Thermo Electron Corporation, Vantaa, Finland). The assessment of absorbance was obtained using the “SkanIt” RE 5.0 for Windows v.2.6 (Thermo Labsystems, Philadelphia, PA, USA) and a mathematical regression analysis for each cell line using a Statgraphics software (Statistical Graphics Corp, 2000, Warrenton, VA, USA) was conducted. The IC50 values were calculated from the semi-logarithmic dose–response curve by linear interpolation.
5
Hydrogen Peroxide-Induced Cell Viability Assay
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Cells were seeded in 96-well plates at density of 20,000 cells/cm2 (6440 cells per well). Proliferating or senescent cells (control, siNC, or siPRDX6 transfected as described above) were treated with H2O2 in triplicate in a concentration range 0.025–3.5 mM for proliferating cells and 0.5–10 mM for senescent cells for 24 h. To determine cell viability by the crystal violet assay [54 (link),55 ], the cells were washed twice with 150 μL PBS and then stained in 30 μL 0.5% (w/v) crystal violet in 20% methanol for 15 min. Plates were washed three times with double distilled H2O and left to dry overnight. Crystal violet was solubilized with 75 μL 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 15 min. Absorbance of crystal violet was measured at 595 nm using a microplate reader (Multiskan EX, Thermo Electron Corporation, Waltham, MA). Alternatively, the MTT assay was performed with CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (#G4000, Promega) according to the manufacturer's protocol. Absorbance of the treated samples was expressed as a percentage of absorbance of untreated cells. IC50 values were estimated using nonlinear regression curve fitting in GraphPad Prism version 8.0.0.
6
MTT Assay for Cell Viability
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Cell lines were treated for 2 days with 10 μg/ml TSA (Sigma) and 0.5 μM ICBP112 (Sigma) which have been dissolved in dimethylsulfoxide, and subsequently prepared for standardized MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; obtained from Sigma) assays. The measurement was performed twice in triplicates. The absorbance was determined at 540 nm and at 620 nm as background control using ELISA reader Multiskan EX (Thermo Electron, Vantaa, Finland).
7
Etoposide Cytotoxicity Assay in HL Cell Lines
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HL cell lines were transfected as indicated and and treated for 20 h with 10 μg/ml Etoposide (Sigma) which has been dissolved in dimethylsulfoxide, and subsequently prepared for standardized MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; obtained from Sigma) assays. The measurement was performed twice in triplicates. The absorbance was determined at 540 nm and at 620 nm as background control using ELISA reader Multiskan EX (Thermo Electron, Vantaa, Finland).
8
Cell Viability Assay with WST-1
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After the removal of the supernatants, cells were washed with Dulbecco’s phosphate buffered saline (DPBS) (Lonza), supplemented with glutamax and antibiotic/antimycotic solution. Cytotoxicity was measured by adding 150 µL of a 1/10 dilution of 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1, Roche, Basel, Switzerland) reagent in serum-free medium to each well. Metabolically active cells convert WST-1 reagent to a formazan that can be measured spectrophotometrically. Formazan formation was determined by reading the plate at 450 nm (Multiskan Ex, Thermo Electron Corporation, Waltham, MA, USA) immediately after WST-1 addition and again after an incubation period of 1 h at 37 °C. The increase in absorbance at 450 nm is proportional to formazan formation. The level of fomazan formed is directly proportional to cell viability.
9
Colorimetric MTT-based Cell Viability Assay
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Evaluation of cell viability was performed by using colorimetric MTT-assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma). The amount of formazan product correlates with the quantity of live cells. Cells were seeded in 24-well plates and were treated with rapamycin or pp242. After 24 h treatment MTT (0.5 mg/ml dissolved in PBS) was added, and cells were incubated for 1.5 h at 37 °C in 5% CO2. The resulting formazan precipitate was dissolved in DMSO (Sigma), gently pipetted and dispensed into 96-well plate. The absorbance was measured at 570 nm wavelength using Multiskan EX (Thermo Electron, Helicon, St. Petersburg, Russia). Each experiment was repeated six times followed by calculation of the standard error of the mean.
10
Quantitative Immunoassay for Viral Antibodies
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At the end of the incubation period, the plates were rinsed three times with Washing Buffer and then treated with 100 μl of 2,2′-azino-bis 3-ethylbenzthiazoline- 6-sulfonic acid (ABTS) (Sigma-Aldrich, Milan) in buffer solution (9.1 mM ABTS; pH 5.0), which reacted with the peroxidase enzyme to yield the color reaction. The plate was then read spectrophotometrically (Thermo Electron Corp., model Multiskan EX, Finland) at a wavelength (λ) of 405 nm. This optical density (OD) reading reflected the extent of immunocomplexes formed by the presence of specific antibodies, which bound to the SV40 synthetic peptide/epitopes/mimotopes. The three SV40 negative control sera were selected from samples below the cut-off value determined with second–degree polynomial regression by plotting the ranked net OD individual values for each peptide. A tendency curve was drawn from a second–degree polynomial regression for Tag A peptide and Tag D peptide, as published for MCPyV and BKPyV virus-like particles (VLPs) (Touzé et al., 2010 (link)). Our OD data/analysis revealed an inflection point corresponding to 0.18 for each peptide (Tognon et al., 2016 (link)).
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