Cell viability was analyzed by a colorimetric assay using the yellow tetrazolium salt 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) that is reduced by viable cells to a purple formazan. A HaCaT cell suspension with 0.1 × 106 cells/mL was prepared and 100 µL of the suspension were added per well of a 96 well plate. After incubation for 48 h, the cells were exposed to the test compounds in decreasing concentration for 1 h and 3 h, respectively. Afterwards, cells were washed with PBS/HBSS (ccPro GmbH, Oberdorla, Germany) and 100 µL MTT solution (0.5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) per well were added and incubation was continued for 3 h at 37 °C. The supernatant was decanted and for formazan solubilization, 100 µL MTT-elution solution (2-Propanol/HCl) (both Carl Roth GmbH + Co. KG, Karlsruhe, Germany) was added per well. The 96 well plate was incubated protected from light for 15 min while shaking. Absorbance was measured with a microplate reader (BioTek PowerWave XS, Agilent Technologies, Santa Clara, CA, USA) at 550 nm and 620 nm. Sodium dodecyl sulfate (SDS, 0.08%) (AppliChem GmbH, Darmstadt, Germany) served as a positive control.
Biotek powerwave xs
Manufactured by Agilent Technologies
Sourced in United States
The BioTek PowerWave XS is a microplate spectrophotometer designed for absorbance-based assays. It provides precise and reliable absorbance measurements across a wide range of wavelengths, enabling users to perform various colorimetric and fluorometric assays in a 96-well microplate format.
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Lab products found in correlation
Mtt solution, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Biosharp (1 mentions)
Uv 2550, by Shimadzu (1 mentions)
Tmb single solution, by Thermo Fisher Scientific (1 mentions)
Vortemp 56 evc, by Labnet (1 mentions)
Peg 6000, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Antioxidant assay kit, by Cayman Chemical (1 mentions)
14 protocols using biotek powerwave xs
1
MTT Assay for Cell Viability Analysis
2
Cell Viability Determination with CCK-8
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Cell viability was tested using a Cell Counting Kit-8 (CCK-8; Biosharp, Hefei City, China). CCK-8 reagent was added to each well, followed by incubation for 2 hours, and the absorbance (optical density) was determined at 450 nm using a fluorescence plate reader (BioTek Power Wave XS; Agilent, Santa Clara, CA, USA). Relative cell viability was determined by the optical density ratio of treated cells over the control.
3
Characterizing Thai Arabica Coffee Bioactives
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Roasted arabica coffee beans originating from Mae Sot District, Tak Province, Thailand were purchased from Doi Muser coffee shop (Tambon Dan Mae Lamoh, Mae Sot District). To prepare the SCGs, 10 g of crushed coffee beans was dripped using a drip coffee bag and 150 mL of hot water (92–96 °C) was added. wSCGs were then used immediately. dSCGs were dried in a hot air oven at 60 °C for 16 h. Five concentrations (0 (control), 5, 10, 25, and 50 g/L) of dry weight equilibrium of wSCGs and dSCGs were prepared for bioassay.
The concentrations of chemical compounds in the wSCGs and dSCGs were analyzed by triple readings in each sample of three replicates as follows. Total phenolic compounds were determined using a microplate reader (Biotek PowerWave XS; Agilent Technologies, Santa Clara, CA, USA) according to the Folin–Ciocalteu procedure [22 (link)]. The total flavonoid compounds were determined by aluminum chloride assay using a spectrophotometer (Shimadzu UV-2550; SHIMADZU, Tokyo, Japan) [23 ]. High-performance liquid chromatography was used to evaluate compounds, such as caffeic acid, coumaric acid, ferulic acid, gallic acid, protocatechuic acid, sinapic acid, vanillic acid, and caffeine as described previously [24 (link)]. The concentrations were calculated based on dry weight.
The concentrations of chemical compounds in the wSCGs and dSCGs were analyzed by triple readings in each sample of three replicates as follows. Total phenolic compounds were determined using a microplate reader (Biotek PowerWave XS; Agilent Technologies, Santa Clara, CA, USA) according to the Folin–Ciocalteu procedure [22 (link)]. The total flavonoid compounds were determined by aluminum chloride assay using a spectrophotometer (Shimadzu UV-2550; SHIMADZU, Tokyo, Japan) [23 ]. High-performance liquid chromatography was used to evaluate compounds, such as caffeic acid, coumaric acid, ferulic acid, gallic acid, protocatechuic acid, sinapic acid, vanillic acid, and caffeine as described previously [24 (link)]. The concentrations were calculated based on dry weight.
4
Quantifying Chlorogenic Acid Content
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TPCs were determined by the Folin–Ciocalteu colourimetric method [20 ]. The reduction reaction was carried out in 210 μL total volume in 96-well microplates. Ten μL of the sample was added to 150 μL of Folin–Ciocalteu reagent (diluted 1:14, v/v in Milli-Q water). After exactly 3 min, 50 μL of Na2CO3 (20%, w/v) was added into each well. Absorbance was recorded at 750 nm using a microplate reader BioTek PowerWave™ XS (BioTek Instruments, Winooski, VT, USA). Calibration curves were constructed using standard solutions of CGA (0.1–1 mg L−1), and results were expressed as μg CGA equivalents mg−1 (μg CGAE mg−1).
5
ELISA Protocol for Protein Quantification
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ELISA kit were purchased from R&D systems (R&D Systems Inc. Minneapolis, MN) and the protocol was conducted according to the manufacturer. In short, capture antibodies were diluted in PBS and added to high-binding 96-well plates 1 day in advance of the experiment. Plates were blocked with reagent diluent (1% BSA in PBS) and washed with washing buffer (0.05% Tween 20 in PBS). Substrate solution was purchased from Life Technologies (TMB Single solution, Thermo Fisher Scientific Inc.), and H2SO4 was used as stop solution. Absorbance was measured at 450 and 570 nm with BioTek PowerWave XS (BioTek Instruments Inc.).
6
Antioxidant Evaluation of Verbascoside
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The antioxidant properties of verbascoside were determined by the method described by Berrington and Lall (2012) [32 (link)]. Stock solutions of verbascoside and ascorbic acid (positive control) were prepared at 10 mg/mL and 2 mg/mL in 100% ethanol, respectively. Twenty microliters of verbascoside stock solution was added to the top well of a 96-well plate and serially diluted to concentrations that ranged from 3.90–500 µg/mL. Ascorbic acid was serially diluted to a concentration range of 0.78–100 µg/mL. Ethanol (10%) was used as a blank. Ninety microliters of a 0.04 M DPPH (2,2-diphenyl-1-picrylhydrazyl) in ethanol were added to each well. The plates were incubated for 30 min and covered in a layer of foil. Color controls (negative controls) were prepared in the exact same manner as above, but distilled water was added to each well instead of DPPH. The absorbances were measured at a wavelength of 515 nm using a BIO-TEK Power-Wave XS multiplate reader (BIO-TEK Instruments, Winooski, VT, USA). From the absorbances an IC50 value was determined. All concentrations of verbascoside and ascorbic acid were tested in triplicate.
7
Evaluating Nanoparticle Surface Hydrophobicity
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The surface hydrophobicity of the different nanoparticles was evaluated by the Rose Bengal method27 . Briefly, 500 μL of nanoparticle dispersions in water (from 0.04 to 4 mg/mL) were mixed with 1 mL of an aqueous solution of Rose Bengal (100 μg/mL). The samples were maintained for 30 min at 25 °C under constant shaking at 1500 rpm (Labnet VorTemp 56 EVC, Labnet International, Inc., Edison NJ, USA). Subsequently, the samples were centrifuged and the amount of Rose Bengal in the supernatants (unbound) was quantified at 548 nm in a microplate reader (BioTek PowerWave XS, BioTek Instruments Inc., Winooski, VT, USA). The absorption of the Rosa Bengal to nanoparticle surface (bound) was determined by the difference among the initial amount of the dye and amount or Rose Bengal unbound. The partitioning quotient (PQ) in Eq. (2) of each sample was plotted versus the total surface area (TSA). The slope of the line of the graph represents the hydrophobicity of the formulation. The higher the slope, the higher the hydrophobicity.
8
MTT Assay for Cell Viability
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The effect of each sample on cell viability was evaluated according to the MTT assay as previously described [44 (link)]. For this assay, 100 μL of cell suspensions (1.5 × 105 cells/mL) were plated in 96-well plates and allowed to stabilize overnight at 37 °C under a humidified atmosphere with 5% CO2. The effect of the vehicle solvent (DMSO) was evaluated in all experiments by exposing untreated control cells to the maximum concentration (0.1%) of DMSO used in each assay. A stock solution of the studied samples was prepared in DMSO and kept at −20 °C, and appropriate dilutions of each sample were freshly prepared just prior to every assay. Cells were then exposed to the respective samples, with a maximal solvent concentration of 0.1% DMSO, and incubated for 48 h. Afterwards, the medium was discarded, and the cells were washed with PBS prior to the addition of MTT solution at 0.45 mg/mL. The crystals of formazan were then allowed to form for 1.5 h and subsequently solubilized with DMSO prior to recording the absorbance at 570 nm on a standard spectrophotometer (Biotek PowerWave XS, BioTek Instruments Inc., Winooski, VT, USA). The results were expressed relative to untreated cells’ viability.
9
Esterase Activity Determination and Thermal Stability
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Esterase activity was determined with p-nitrophenyl butyrate (pNPB) as a substrate in a microplate format (BioTek PowerWave XS, BioTek Instruments Inc., Winooski, USA) (Billig et al. [2010 (link)]). To avoid the adsorption of proteins to the plastic vials, the dilution was carried out in the presence of 15% poly(ethylene glycol) (PEG6000, Sigma-Aldrich Co., St. Louis, USA) in Davies buffer (Davies [1959 (link)]) between pH 6.5 and 9.5 or in 100 mM Tris-HCl. One unit of esterase activity was defined as the amount of enzyme required to hydrolyze 1 μmol pNPB per min (Alisch et al. [2004 (link)]). To investigate their thermal stability, 250 μg/mL of enzymes were incubated in 100 mM Tris buffer (pH 8.5) at 50°C, 55°C and 60°C for up to 1 h. Residual esterase activity against pNPB was determined at 25°C in triplicate. The Michaelis-Menten kinetic constants for the hydrolysis of pNPB by Tcur1278 and Tcur0390 were determined at 25°C and pH 8.5.
10
Cytotoxicity of Thiostrepton on Breast Cancer Cells
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The cell viability MTT assay was used to measure the cytotoxic effect of thiostrepton on MCF-7 or MDA-MB-436 cells after treatment for 24, 48 and 72 h. In brief, cells were seeded in a 96-well plate at a density of 2,000 cells/well. After 24 h, the culture medium was replaced with various concentrations of thiostrepton. At each time-point, the viability of the tested cells was determined by adding MTT (Sigma-Aldrich; Merck KGaA) and incubating at 37°C, allowing formazan crystals to form. The crystals were dissolved in dimethyl sulfoxide (Merck KGaA) and the absorbance at 490 nm was measured with a microplate reader (BioTek PowerWave XS; BioTek Instruments, Inc.). Five replicate wells were used for each analysis, and three independent experiments were performed.
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