Cell viability of adherent cells was measured by WST-1 proliferation assay (Roche, 11644807001) or CellTiter- Glo Luminescent cell viability assay (Promega, Madison, WI, USA) for GSCs, using GloMax Explorer instrument (Promega, G9242), according to the manufacturer’s instructions. GSC spheroids were analyzed by scanning the wells in InCell Analyzer 2200 (GE), stitching the pictures to obtain full-well images, and measuring the spheroids’ size using Fiji Image J software (NIH, open source). Generally, the experiments were repeated at least three times for each experimental condition.
Wst 1 proliferation assay
Manufactured by Roche
Sourced in Germany, Switzerland, United States
The WST-1 proliferation assay is a colorimetric assay used to measure cell viability and proliferation. It utilizes the tetrazolium salt WST-1, which is cleaved by cellular enzymes, resulting in the formation of a colored formazan dye. The amount of formazan dye produced is directly proportional to the number of metabolically active cells in the sample, allowing for the quantification of cell proliferation or cytotoxicity.
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Lab products found in correlation
In cell analyzer 2200, by GE Healthcare (1 mentions)
Glomax explorer instrument, by Promega (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Epoch 2 microplate spectrophotometer, by Agilent Technologies (1 mentions)
Temozolomide, by Santa Cruz Biotechnology (1 mentions)
Spss statistics 22, by IBM (1 mentions)
Pen strep glutamine, by Thermo Fisher Scientific (1 mentions)
Brivanib, by Selleck Chemicals (1 mentions)
Azd4547, by Selleck Chemicals (1 mentions)
Bio tek elisa reader el 800, by Agilent Technologies (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Afatinib, by Calibre Scientific (1 mentions)
Recombinant areg, by R&D Systems (1 mentions)
Recombinant hb egf, by R&D Systems (1 mentions)
Trastuzumab, by Roche (1 mentions)
Cetuximab, by Merck Group (1 mentions)
Wst 1 proliferation reagent, by Roche (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Spectramax plus, by Molecular Devices (1 mentions)
Elisa reader, by Agilent Technologies (1 mentions)
20 protocols using wst 1 proliferation assay
1
Quantifying Adherent Cell Viability
2
Cell Viability Assessment by WST-1 Assay
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The viability of the cells was examined using the WST-1 proliferation assay (Roche, Basel, Switzerland). This assay is based on the measurement of biochemical activity relative to cellular respiratory processes. A substrate (tetrazole salt) is biotransformed by mitochondrial dehydrogenase into soluble formazan and its concentrations then measured spectrophotometrically at 450 nm. Next, 5 μl of the WST-1 solution was dispensed into each cell culture well. The plates were incubated for 2 h at 37 °C. The absorbance at 450 nm was measured using a BioTek Synergy multiplate reader (BioTek, Winooski, VT, USA). The obtained results were normalized to measurements from non-treated cells and are represented as the percent growth inhibition. The drug IC50 values were calculated for the healthy donors using GraphPad Prism (GraphPad Software, La Jolla, CA, USA), by plotting the percent viability against the drug concentration and fitting the curve using an allosteric sigmoidal model. The PBMC-growth inhibition was calculated at the determined IC50 concentration using an allosteric sigmoidal curve fit. One-way analysis of variance (ANOVA) with Tukey post hoc and t-tests were used for determining the significance of differences between groups.
3
Fibroblast Proliferation Assay with WZB117
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Proliferation was measured by WST-1 Proliferation Assay (11644807001, Roche, Basel, Switzerland). Fibroblasts were seeded at 3 × 103 cells/well (100 μL) in 96-well plates in DMEM with 2% FBS overnight. Fibroblasts were treated with WZB117 at indicated concentration in <2% FBS DMEM and determined at indicated time points. The cell medium was replaced with the WST-1 reagent and then incubated at 37 °C for 2 h. Absorbance was monitored with an EpochTM 2 Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at 450 nm with a reference wavelength of 620 nm.
4
Dose-Response Synergy Analysis of Anticancer Agents
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Cells were grown, synchronized by reduced serum supply and seeded in quintuplets in 96 well plates. Cells were synchronously treated with increasing concentrations of Siomycin A, everolimus, cisplatin (Charité Berlin dispensary) or temozolomide (Santa Cruz Biotechnology Inc.) and 0.1% DSMO, respectively, to obtain dose response curves as recommended by the Chou-Talalay method [35 (link)]. Additionally, several non-constant combinations of >IC50 concentrations of siomycin A or everolimus and cisplatin or temozolomide were assessed. WST-1 proliferation assay (Roche; Basel, Switzerland) was performed colorimetrically quantified. Data was analyzed by CompuSyn 1.0 at Fa=0.5 [63 ] and IBM SPSS Statistics 22 software.
5
Evaluating FGFR Inhibitors on Gastric Cancer Cells
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KatoIII cells (HTB-103, ATCC) and AGS cells (CRL-1739, ATCC) were grown in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum and 100 U/mL of Pen Strep Glutamine (Gibco). All cells were cultured at 37°C in a humidified atmosphere and 5% CO2. Survival of KatoIII and AGS cells was determined using the WST-1 Proliferation Assay (Roche). We tested multiple FGFR inhibitors including TKI-258, Brivanib (BMS-540215), Ponatnib (AP24534), and AZD4547 (Selleck Chemical). Cells were seeded at a density of 2 × 104 cells/well in 96-well microtiter plates, 100 μL medium/well and maintained 18 h for attachment. Afterwards, we treated the cultures with varying concentrations of each drug diluted in DMSO. After 30 h incubation, 10 μL of WST-1 reagent was added followed by 1 h at 37°C. The cleavage of tetrazolium salt (WST-1) into a visible formazan by viable cells was spectrophotometrically measured using a reference wavelength of 450 nm. Each test was performed in triplicate. Percentages of cell survival were calculated as follows:% cell survival = (absorbance of treated cells/ absorbance of cells with vehicle solvent) × 100. The half inhibitory concentration (IC50) was calculated with a non-linear regression from the dose–response curve.
6
Comparative Viability Assay for Pancreatic and Breast Cancer Cells
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Viability/proliferation of 8932 pancreatic cancer cells and MCF-7 breast cancer cells was evaluated using a WST-1 proliferation assay (Roche Diagnostics). For quantification 2.5 x 103 cells were seeded in triplicates in 96-well plates and were left to adhere overnight. Metabolic activity of cells, indicative of cell proliferation/viability, was assessed after addition of 10 μl (per 100 μl medium) of a liquid reagent containing tetrazolium salt to the respective cell medium. Changes in formazan dye generation were measured in an ELISA reader after 30 min of incubation at 37°C and 5% CO2 (Bio-Tek ELISA Reader EL 800, Bio-Tek Instruments GmbH, Bad Friedrichshall, Germany). Results of absorbance measurements (450 nm) of both cell lines were plotted in one graph to display relative metabolic activity of both cell lines.
7
HUVECs Proliferation Assay
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HUVECs were seeded in 96-well cell culture plates and transfected with siRNA/pre-miR 1 day after seeding. Proliferative capacity in siRNA/pre-miR-modulated cells, a WST-1 proliferation assay (Roche, Germany) was applied. Twenty-four hours after transfection medium was replaced by a starvation medium containing 0, 1% FCS, and without supplements for additional 24 h. Forty-eight hours after transfection medium was changed and replaced by medium containing WST-1 reagent according to the manufacturer’s instructions. Then, WST-1 absorbance was measured at 450 nm.
8
Cell Proliferation Assay using WST-1
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Cell proliferation was measured using a WST-1 proliferation assay (Roche, Basel, Switzerland) according to manufacturer’s instructions. In brief, sextuplicates of each cell line (1 × 104 cells/well) were seeded in microtiter plates in 100 µl medium in a humidified atmosphere (37 °C, 5% CO2) for 24 h. Then, 10 µl/well Cell Proliferation Reagent WST-1 were added and incubated for another 2 h. Before measurement, plates were shaken for 1 min and OD/absorbance was determined at 450 nm with 690 nm as reference against blank as background control on a microtiter plate reader (Infinite 200 pro, Tecan).
9
Cell Proliferation Assay with Targeted Therapies
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The water-soluble tetrazolium (WST-1) proliferation assay (Roche Diagnostics) was used to measure cell proliferation after knockdown or stimulation as described earlier [17 (link)]. Cells were treated with cetuximab (1/10 µg/ml, Merck), trastuzumab (5/20 µg/ml, Roche), afatinib (0.5 µM, Biozol), DMSO (0.05%, afatinib solvent), trastuzumab solvent (described in [17 (link)]) or cetuximab solvent (8.48 mg/ml NaCl, 1.88 mg/ml Na2HPO4 × 7H2O, 0.41 mg/ml NaH2PO4xH2O) for 72 h (cell numbers see Table S1, Additional file 1 ). In case of stimulation, cells were treated with 5 ng/ml recombinant HBEGF or 15 ng/ml recombinant AREG (R&D Systems).
10
Investigating SOX9 Knockdown Effects on HepG2 Cell Growth
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To test whether loss of function of SOX9 expression influence cellular growth rates of HepG2 cells, we performed SOX9 knock-down experiments. HepG2 cells were transiently transfected with short interfering RNA (siRNA) to knock-down SOX9 (SOX9; 20 nM, Hs_SOX9_3 FlexiTube siRNA, cat. No. SI00007609, Qiagen) using fast forward transfection procedure according to HiPerFect Transfection Reagent (Qiagen) protocol. As a reference, the AllStars Negative Control (20nM, Qiagen) was used.
After transfection, we measured the cellular growth rate at every 24 hours over a period of time of 96 hours by applying the WST-1 proliferation assay (Roche Applied Science, Mannheim, Germany). In detail, after standard trypsinisation, 5000 HCC cells per well were seeded in a 96-well culture plate. After transfection with a siRNA against SOX9 or respective control (AllStars Negative Control, Qiagen), cells were incubated in 100 μl of normal growth medium for 96 h and the WST-1 proliferation reagent (Roche Applied Science) was added every 24 hours according to manufacturer’s recommendations. At each time point, after 3 hours WST-1 incubation colorimetric changes were measured using a SpectraMax Plus (Molecular Devices) at a wavelength of 450 nm with a reference wavelength at 620 nm.
After transfection, we measured the cellular growth rate at every 24 hours over a period of time of 96 hours by applying the WST-1 proliferation assay (Roche Applied Science, Mannheim, Germany). In detail, after standard trypsinisation, 5000 HCC cells per well were seeded in a 96-well culture plate. After transfection with a siRNA against SOX9 or respective control (AllStars Negative Control, Qiagen), cells were incubated in 100 μl of normal growth medium for 96 h and the WST-1 proliferation reagent (Roche Applied Science) was added every 24 hours according to manufacturer’s recommendations. At each time point, after 3 hours WST-1 incubation colorimetric changes were measured using a SpectraMax Plus (Molecular Devices) at a wavelength of 450 nm with a reference wavelength at 620 nm.
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