Cell proliferation assays were performed using Cell Proliferation Reagent WST-1 (Millipore Sigma, 11644807001) according to the manufacturer’s protocol. For colony formation at low density on plastic, 500 cells/well were plated into 6-well plates. Cultures were maintained for 14 d to allow the formation of cell colonies, and colonies were stained with 1% Crystal Violet and manually counted. For cell apoptosis assays, 105 cells were stained with Annexin V-FITC/PI solution using Dead Cell Apoptosis Kit (Thermo Fisher Scientific, V13242) according to manufacturer’s instructions and subjected to flow cytometry analysis (BD FACSAria II, BD Biocsience) to detect apoptotic cells.
Cell proliferation reagent wst 1
Manufactured by Merck Group
Sourced in United States, Germany
The Cell Proliferation Reagent WST-1 is a colorimetric assay for the quantification of cell proliferation and viability. It measures the metabolic activity of cells by detecting the reduction of a tetrazolium salt (WST-1) to a colored formazan product. The intensity of the color is proportional to the number of viable cells in the sample.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Trypan blue dye, by Merck Group (3 mentions)
Imark microplate reader, by Bio-Rad (3 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Multiskan sky, by Thermo Fisher Scientific (3 mentions)
Epoch microplate spectrometer, by Agilent Technologies (2 mentions)
Beta galactosidase staining kit, by Takara Bio (2 mentions)
Chariot protein delivery reagent, by Active Motif (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Synergy h4 hybrid reader, by Agilent Technologies (1 mentions)
Victor3 1420 multilabel counter, by PerkinElmer (1 mentions)
Cy5 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Fujifilm (1 mentions)
Phosphate buffered saline pbs, by Fujifilm (1 mentions)
Imark plate reader, by Bio-Rad (1 mentions)
Infinite 200, by Tecan (1 mentions)
Wst 1, by Merck Group (1 mentions)
73 protocols using cell proliferation reagent wst 1
1
Proliferation, Colony, and Apoptosis Assays
2
WST-1 Assay for Cell Proliferation
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Cell proliferation was determined by using WST-1 Cell Proliferation Assay. Cell Proliferation Reagent WST-1 was purchased from MilliporeSigma. 2 × 104 FLO-1 cells were seeded in a 96-well plate. When cells reached 50–60% confluence, different concentrations of carnosol or vehicle controls were added to each well. After cells were cultured for 24 h, 10 μl WST-1 reagent was added to each well and cells were cultured for additional 1 h. Then the absorbance of each well was determined by using a BioTek Synergy H4 hybrid reader at 440 nm. Medium without cells was used as a blank. Carnosol was dissolved in ethanol and the same concentrations of ethanol were used in the control groups. The experiments were repeated three times for the experiments of siRNA, N-acetyl cysteine and carnosol’s effects on cell proliferation and twelve times for apocynin experiments.
3
Evaluating THP-1 Cell Viability and Macrophage Killing
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Cell viability of THP-1 cells after exposure to drugs was determined using Cell Proliferation Reagent, WST-1 as per manufacturer’s recommendation (Sigma-Aldrich, St. Louis, MO, United States). For macrophage killing experiments, THP-1 cells were infected with single-cell bacterial suspensions as previously described (Mawatwal et al., 2017 (link)). After 4 h post-infection, the extracellular bacteria were removed by overlaying macrophages with RPMI medium containing 200 μg/ml of amikacin. After 2 h of incubation, cells were washed and infected macrophages were overlaid with RPMI medium containing drugs for indicated time points. In another experiment, infected macrophages were pre-treated for 1 h with 3-methyl adenine (3-MA, 10 mM), a selective PI3K inhibitor that inhibits autophagy before treating with NSC 18725 for varied time points. Co-localization experiments were performed by infecting THP-1 cells with GFP labeled M. bovis BCG at a MOI of 1:10 as described above followed by treatment with NSC 18725 treatment for 12 h. For bacterial enumeration, 10-fold serial dilutions were prepared and plated on MB7H11 plates at 37°C for 3–4 weeks.
4
Silver Nanoparticles Cell Viability Assay
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Viability of cells treated with silver nanoparticles was measured using by the Cell Proliferation Reagent WST-1 (Sigma Aldrich) in accordance with the manufacturer's instructions. Briefly, PC3-PIP and PC3-FLU cells (2 × 103 cells per well) were cultured in 96 well plate of flat bottom in a final volume of 100 μl per well culture medium. The silver nanoparticle concentration was maintained at 0.5 nM at each well. At the trehalose containing wells, the concentration of trehalose was maintained at 0.75 M. At the indicated time points, 10 μl of this reagent was added to each well and incubated for an additional 1 h. Then, the plate was shaken thoroughly for 1 min. The absorbance of samples was measured under a wavelength of 450 nm using a VICTOR3™ 1420 Multi-label Counter (PerkinElmer, USA). The percent viability of cells was calculated by comparison to that of untreated control cells.
5
Cell Proliferation Assay using WST-1
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The Cell Proliferation Reagent WST-1 (Sigma) is used for the spectrophotometric quantification of cell proliferation.
6
Cell Proliferation and Immunostaining Protocol
7
Antioxidant Activity Assay Protocol
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3-Hydroxytyramine hydrochloride was purchased from Tokyo Chemical Industry (Tokyo, Japan). Ethanol (95%), ammonia water (28%), FeSO4·7H2O, FeCl2·4H2O, and H2O2 were purchased from Kishida Chemical (Osaka, Japan). APF was purchased from Goryo Chemical (Sapporo, Japan). Phosphate buffered saline (PBS) and LPS were purchased from Wako chemical (Osaka, Japan). Cell Proliferation Reagent WST-1 was purchased from Sigma-Aldrich (St. Louis, MO, USA).
8
Cell Viability Assay of Nanotubes
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For the cell viability assay, two sets of Hela cells were grown in a 96-well tissue culture plates for either one or four days in the presence of the nanotubes (20 µg/mL in DMEM at 5% CO2 at 37 °C). In each well, 200 µL of the cell sample was incubated in 20 µL of Cell Proliferation Reagent WST-1 (Sigma Aldrich) for 1.5 h at room temperature to lyse the cells. After the incubation period, the resulting formazan dye was quantitated with a scanning multi-well plate spectrophotometer (Biorad iMark plate reader). The absorbance directly correlates to the number of viable cells. Blank absorption collected only from the same volume of culture media, and WST-1 reagent (in the absence of cells) was subtracted from each absorbance value. Control cells were prepared by following the same protocol, but without SWCNTs administration. The viable cell fractions (percentages) were calculated by normalizing the cell number with nanotube incubation by the control cell number without nanotube incubation.
9
Cell Proliferation Assay in Breast Cancer
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MCF‐7 and MCF‐7 Luc cell suspensions were prepared and added to a 96‐well plate and cultured. The 96‐well cell culture plates were removed from culture at seven different time points (day one to day seven); at each point, the medium in each well was discarded and Cell Proliferation Reagent WST‐1 (Sigma‐Aldrich, USA) (110 μl of a 1:10 dilution of WST‐1) was added. The plates were then placed at 37°C in a 5% CO2 incubator and incubated in the dark for 2 h. After shaking for 10s, the OD value of the cells at 450 nm was then measured with an Infinite 200 multifunctional microplate reader (Tecan Trading AG, Switzerland). The growth curve of MDA‐MB‐231 and MDA‐MB‐231 Luc cells was determined by the same method.
10
Multifunctional Nanoparticles for Targeted Drug Delivery
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The chemicals tetraethyl orthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), safranin O, glutathione (GSH), (3-mercaptopropyl) trimethoxysilane (MPTMS), 2,2′-dipyridyl disulfide, peroxidase from horseradish (HRP), H2O2 30%, β-glycerophosphate, and poly(ethylene glycol) methyl ether thiol (Mn 800) were provided by Aldrich. Doxorubicin hydrochloride (DOX) was supplied by Sequoia Research Products. For cell culture studies, U271 malignant glioma cells, Eagle’s minimum essential medium (MEM), penicillin–streptomycin, phosphate-buffered saline (PBS), fetal bovine serum (FBS), glutamine, trypsin, and cell proliferation reagent (WST-1) were obtained from Sigma-Aldrich. Tyramine-derivatized hyaluronic acid was purchased from Contipro A.S. Ultra-pure chitosan (UP CL214, Protasan) was supplied from Pronova Biomedical (Norway). The analytical-grade solvents were provided from Scharlab (Barcelona, Spain). All reagents were used as received.
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