Cells following 48 h of siRNA transfection or intact cells were seeded in a 96-well plate at 5000 cells per well. After the cells attached to the bottom, a series of doxorubicin concentrations was applied to the wells. For PLD1 inhibitor experiments, a series of doxorubicin concentrations combined with 10 μM of PLD1 selective inhibitor VU0155069 (Santa Cruz Biotechnology) was applied to the wells after 48 h of seeding intact cells. After 48 h or 72 h of treatment, WST-1 assay reagent (Roche Life Science, Indianapolis, IN) was added according to the manufacturer's protocol and incubated for 2 h, followed by detecting the absorption at 450 nm. We measured 3 technical replicates for each of 4 biological repeats for each experimental group.
Wst 1 assay reagent
Manufactured by Roche
Sourced in Switzerland
The WST-1 assay reagent is a colorimetric reagent used for the quantification of cell viability and proliferation. It works by measuring the reduction of a tetrazolium salt into a colored formazan product, which can be detected spectrophotometrically. The WST-1 assay provides a simple, rapid, and sensitive method for analyzing cellular metabolism and growth.
Automatically generated - may contain errors
Lab products found in correlation
Vu0155069, by Santa Cruz Biotechnology (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
Mpr a4i, by Tosoh (1 mentions)
Ix71 epifluorescence microscope, by Olympus (1 mentions)
Countess 2, by Thermo Fisher Scientific (1 mentions)
Hoechst dye, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
8 protocols using wst 1 assay reagent
1
Synergistic Doxorubicin-PLD1 Inhibitor Effects
2
Vipera venom cytotoxicity assay
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The effect of the Trachinus vipera purified venom was determined by the use of WST-1 assay. HCT116 cells were seeded in 96-well plates (5000 cells/well). 24 hours later, cells were treated with different concentrations of the venom (50, 100, 500 and 1000 μg/ml) diluted in phenol red-free media (100 μl). After 24 h, 48 h and 72 h WST-1 assay reagent (Roche Applied Science, Mannheim, Germany) was subsequently added (10 μl) to each well and cells were incubated for 4 hours at 37 °C before the absorbance lecture. Each well was measured at the wavelength of 450 nm and reference wavelength of 690 nm, using a scanning multiwell spectrophotometer (Synergy 2). Statistics were calculated using Student’s t-test assuming unequal variances and the mean ± SEM is presented. Each experiment was performed in triplicate and repeated three times.
3
Antiproliferative Effects of Estrogen and Tamoxifen
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The effect of 17β-estradiol (E2) and 4-OH-tamoxifen was determined by use of WST-1 assay. T47D cells were seeded at a density of 400 000 cells in a 60 mm Ø culture dish (28.3 cm2) in PEST-free media and transfected the following day as described. Forty-eight hours after transfection, cells were re-seeded in phenol red-free DMEM supplemented with 5% charcoal stripped serum in a 96-well plate (5000 cells/well). After an additional 24 hours, cells were incubated at 37°C in phenol red-free DMEM supplemented with 1% charcoal stripped serum with either control treatment (EtOH), 1 nM 17β-estradiol (E2) (Sigma #E2758, Sigma-Aldrich Co, St. Louis, MO) or 1 nM E2 and increasing concentrations of 4-OH-tamoxifen (10 nM, 100 nM and 1 μM) (Sigma #H7904, Sigma-Aldrich Co), the active metabolite of tamoxifen, for 4 days. WST-1 assay reagent (Roche Applied Science, Mannheim, Germany) was subsequently added (10 μl) to each well and cells were incubated for 4 hours at 37°C before the absorbance of each well was measured at the wavelength of 450 nm and reference wavelength of 690 nm, using a scanning multiwell spectrophotometer (Synergy 2). Statistics were calculated using Student’s t-test assuming unequal variances and the mean ± SD (standard deviation) is presented. Each experiment was measured in triplicate and repeated five times.
4
Cell Viability Assay Protocol
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Cells were seeded at 5 × 102 cells/well in 96-well plates, and then incubated for the indicated times. At the end of time points, cells were incubated with 10 μl per well of WST-1 assay reagent (Roche). After 30-min incubation, absorbance was measured at 450 nm. Each experiment was repeated three times.
5
Cell Proliferation Assay by WST-1 Reagent
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Cell proliferation was analyzed by WST-1 assay reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. Raw264.7 cells were cultured on 96-well plates (Nunc; Thermo Fisher Scientific, Waltham, MA, USA) in 100 µl complete medium containing with or without 100 ng/ml rmSCRG1. After five days, the cells were added with 10 µl WST-1 reagent and incubated for 1 h. The absorbance was measured using an MPR-A4i microplate reader (Tosoh Corp., Tokyo, Japan) at 450 nm.
6
Enzalutamide Sensitivity in Prostate Cancer
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LNCaP95 control and LNCaP95-Enza-R cells were cultured in media containing DMSO (Sigma) or 50 μM enzalutamide for at least one week. Cells were counted using the Countess II (Life Technologies). Average cell number/mL and standard error of the mean (SEM) were calculated from triplicate wells. For LNCaP95 control an Enza-R cells stably expressing shRNAs, cells were treated with DMSO or 50 μM of enzalutamide and counted as above. For LNCaP95 Snail expressing cells, ethanol (EtOH) or 4OHT pre-treated LNCaP95 cells were treated with DMSO or 6.25, 12.5, 25 and 50 μM of enzalutamide. Cell viability was measured after 10 days of drug treatment using the WST-1 assay reagent (Roche). For LNCaP95 Snail cells stably expressing shRNAs, cells were treated with DMSO or 50 μM of enzalutamide for one week. Cells were then fixed in 4% paraformaldehyde (PFA, Sigma) and stained with Hoechst dye (Sigma). Wells were then imaged on an inverted Olympus IX 71 epifluorescence microscope. Total area was calculated from three representative images per well using ImageJ (Version 2.0.0-rc-41/1.50d).
7
Macrophage and Splenocyte Viability Assay
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RAW 264.7 macrophages or splenocytes were seeded onto 96‐well polystyrene plates. Cells were stimulated as indicated or cultured as unstimulated control. After the indicated time‐points, viable cells were quantified using WST‐1 assay reagent (Roche Diagnostics) according to the manufacturer's instructions.
8
Cell Viability Assay with pTRAP150
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SiHa cells were seeded in 96-well plates at a density of 4 ×103, 8 × 103 or 16 × 103 cells/well. Cells were transfected the following day, either with control vector (pUC19) or with increasing concentrations of pTRAP150. Cell viability was measured at 24 or 48 h post-transfection. Cell culture medium was replaced with fresh growth medium at 2 h prior to the WST-1 assay. WST-1 assay reagent (Roche) was added to a final concentration of 10% of the cell culture medium followed by incubation for 0.5–4 h at 37°C in 5% CO2. The absorbance of the formazan product was measured at 450 nm using a TriStar Plate Reader (Berthold Tech).
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