CyQUANT® cell proliferation assay kit (ThermoFisher Scientific) was used to measure DNA concentration. Briefly, the lysed cell supernatant was supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA, Sigma Aldrich) to prevent DNA degradation. In a 96 well plate, 100 μl of standards, samples, or blank and 100 μl of CyQUANT® GR dye was added and incubated for 5 min at room temperature. The fluorescence of the samples (480/520 λex/λem) was measured using EnSightTM multimode plate reader (PerkinElmer, Inc.). Bacteriophage λ DNA was used to make a standard curve ranging from 0 to 1000 ng/ml.
Ensight
Manufactured by PerkinElmer
Sourced in United States, United Kingdom, Singapore
The EnSight is a multimode plate reader that offers high-performance detection capabilities for a wide range of applications in life science research. It provides sensitive and accurate measurements of various biochemical and cellular assays, including luminescence, fluorescence, and absorbance.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo, by Promega (3 mentions)
Multidrop combi, by Thermo Fisher Scientific (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
On targetplus sirna library, by Horizon Discovery (2 mentions)
Cck 8 reagent, by Wuhan Servicebio Technology (2 mentions)
Steady glo luciferase assay system, by Promega (2 mentions)
Cell proliferation kit 2, by Roche (2 mentions)
Cck 8, by AbMole (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
384 well tissue culture plate, by Corning (2 mentions)
Echo 550, by Beckman Coulter (2 mentions)
Nano glo luciferase assay, by Promega (2 mentions)
96 well clear flat bottom, by Greiner (2 mentions)
Uv transparent microplates, by Tecan (2 mentions)
Infinite m200 pro microplate reader, by PerkinElmer (2 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
115 protocols using ensight
1
DNA Quantification using CyQUANT Assay
2
Quantification of IL-8 in Cell Culture
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IL-8 in cell culture supernatants after the stimulation of cells was detected by a CXCL8/IL-8 DuoSet ELISA Kit (R&D Systems; Minneapolis, MN, USA) according to manufacturer’s instruction. Briefly, MaxiSorp 96-well plates (ThermoScientific; Waltham, MA, USA) were pre-coated with the capture antibody (4 μg/ml in PBS), incubated for 2 h at rt, and washed four times with 0.05% Tween 20 (Sigma-Aldrich) in PBS (v/v) using a HydroSpeedTM washer (Tecan Group Ltd; Männedorf, Switzerland). Blocking was performed with 1% bovine serum albumin (BSA) (Carl Roth; Karlsruhe, Germany) in PBS (w/v) for 1 h at rt. After washing, supernatants or IL-8 standard were added to wells and incubated for 2 h at rt followed by another washing step. The detection antibody was diluted to 20 ng/ml with 0.1% BSA/0.05% Tween 20 in PBS (w/v/v), added to the plate and incubated for 1 h at rt. After another washing step, streptavidin-HRP was added and incubated for 30 min at rt, followed by a final washing step. The substrate OPD (ThermoScientific) was added and incubated at 37 °C in the dark until the color development. Absorbance was periodically measured using a multimode plate reader EnSightTM (Perkin Elmer; Waltham, MA, USA) at 450 nm to avoid values above 0.4. After addition of 1 M sulfuric acid (ThermoScientific) to stop the reaction, final absorbance was measured at 492 nm.
3
DNA Quantification using Fluorescence Assay
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Sample DNA was quantified using a DNA Quantitation Kit Fluorescence Assay (Sigma-Aldrich, Austria) as per the manufacturers’ instructions. Briefly, 10 μL of the proteinase K digest was mixed with 200 μL of bisbenzimide H33258 dye. Samples were measured in triplicates using a PerkinElmer EnsightTM multimode plate reader.
4
Kynurenine Production Assay for IDO Activity
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IDO activity, represented as concentration of kynurenine produced, was analyzed by a spectrophotometric assay reported earlier31 (link). Cell-free spent culture media were harvested from primary macrophage or macrophage/MSC co-cultures and used immediately for assay. To 120 μl of spent medium, 60 μl of 30% trichloroacetic acid [Sigma Aldrich] was added, mixed thoroughly and centrifuged at 8000 g for 5 min at room temperature. From the clarified supernatant, 85 μl was transferred immediately to 96-well plates and 85 μl of 1% Ehrlich reagent prepared in glacial acetic acid [Sigma Aldrich] was added and incubated for 10 min at room temperature. Absorbance was read at 490 nm on a multimode plate reader [EnsightTM, Perkin Elmer]. Concentrations were determined against a kynurenine standard [Sigma].
5
Colorimetric Quantification of U343MG-BM-MSC Co-culture
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U343MG were co-cultured with BM-MSCs in 24-well transwell chamber (0.4-µm pore size polyester (PET) membrane from BD Biosciences, San Jose, CA, USA). U343MG cells were seeded at the density of 3000 cells and BM-MSCs were seeded at the density of 1000 cells. Cells were co-cultured for 24 or 48 h. At the end of the treatment period, cells in the lower compartment were fixed with p-formaldehyde and staining with crystal violet. After 15 min of incubation, the excess dye was removed by performing three washes with PBS. One hundred microliters of 1% SDS solution were added to each well and was incubated for 1 h at room temperature under gentle stirring. The wells were read at 595 nm with the EnSightTM multimode plate reader (Perkin Elmer, Waltham, MA, USA).
6
Quantifying Glycosaminoglycans in Hydrogels
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For quantification of glycosaminoglycans, a standard protocol based on dimethyl-methylene blue (DMMB) precipitation was performed. Prior to the assay, PEG-dextran hydrogels were digested using dextranase (Cellendes, Germany) while no pretreatment was necessary for fibrin and silk/fibrin hydrogels. Briefly, samples were digested using 30 U/mL proteinase K (Sigma-Aldrich, Austria) at 56°C overnight before addition of DMMB (Sigma-Aldrich, Austria) reagent. Precipitates were centrifuged and the pellet was dissociated using decomplexion solution. Subsequently, absorption was measured at 560 nm within a PerkinElmer EnsightTM multimode plate reader. A standard curve was prepared by the dilution of chondroitin sulfate (Sigma-Aldrich, Austria) in phosphate buffered EDTA/cysteine solution.
7
Ultrasensitive Serum Insulin Quantification
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Insulin concentration in serum after 13 weeks of energy drink/dietary interventions was measured using Ultrasensitive Mouse Insulin ELISA kit (Mercodia, Winston Salem, NC, USA). The procedure conducted was as per the manufacturer’s instructions. Briefly, 25 µL of serum samples were incubated with 100 µL enzyme-conjugated antibody solution for 2 h at room temperature. The samples were subsequently incubated with 200 µL TMB substrate for 15 min, and then the reaction was stopped by adding 50 µL stop solution. Optical density was measured at 450 nm within 30 min after adding the stop solution (EnsightTM, PerkinElmer, Waltham, MA, USA). The results of the standards were used to construct a non-linear log curve, which was then used to interpolate the concentrations of the samples.
8
Colorimetric Serum Lipid Assay
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Serum triglycerides and total cholesterol concentrations were determined by using colorimetric assays according to the manufacturer's instructions (Randox, UK). Briefly, 2 μL of serum was added to 200 μL of reagent and incubated for 10 min at room temperature (RT). Optical density was measured using an EnSightTM multimode plate reader (Perkin Elmer, USA) at a wavelength of 546 nm. Assay standards were used to develop a linear regression line in which sample lipid concentrations were interpolated from.
9
Quantifying ROS Production in Cells
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ROS production evaluation was assessed by using the probe H2DCF‐DA (Molecular Probes, Invitrogen) that under ROS presence is oxidized to fluorescent 2′,7′‐dichlorofluorescein. Cells were seeded in 96‐well plate Lumox® (Sarsteadt, 10,000 cells/well) and maintained in complete culture medium. The day after seeding, cells were treated with IH, IH/N or control protocols in serum‐free medium.
After the protocols, the cells were kept in the dark for 30 min at 37°C in PBS/glucose medium containing 20 μM of H2DCF‐DA. Then, the solution was replaced with fresh PBS/glucose medium; fluorescence intensity was quantified by EnSightTM multimode plate reader (Ex: 485 nm; Em: 520 nm; PerkinElmer Inc.) and normalized to the control.
After the protocols, the cells were kept in the dark for 30 min at 37°C in PBS/glucose medium containing 20 μM of H2DCF‐DA. Then, the solution was replaced with fresh PBS/glucose medium; fluorescence intensity was quantified by EnSightTM multimode plate reader (Ex: 485 nm; Em: 520 nm; PerkinElmer Inc.) and normalized to the control.
10
Quantification of Cellular Glutathione
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The monochlorobimane fluorometric method was used to measure the amount of reduced glutathione (GSH) in cells [45 (link)]. Briefly, cells were grown to confluence in 96-well plates and proteins were extracted in 75 µL of 1% digitonin in 50 mM Tris Assay Buffer (pH 7.4). Samples (50 µL) were mixed with 50 µL of Working Solution (100 µM mCB and 1 U/mL glutathione-S-transferase in 50 mM Tris Assay buffer [pH 7.4]) in a black 96-well plate with a clear bottom. The assay plate was incubated in the dark for 60 min at RT. Fluorescence generated by the GSH-mCB adduct was measured at 485 nm using a microplate reader (ENSIGHT, PerkinElmer) with an excitation wavelength of 380 nm. The GSH content (µM) in samples was extrapolated from a standard curve (0–100 µM of reduced GSH diluted in 50 mM Tris Assay buffer [pH 7.4]) and normalised relative to the total protein content in each well. Data show mean ± SD from n = 3 experiments, each performed in duplicates.
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