Wst 8 cell proliferation assay kit

Manufactured by Cayman Chemical

Sourced in United States

The WST-8 Cell Proliferation Assay Kit is a colorimetric assay used to measure cell proliferation and cytotoxicity. It utilizes the water-soluble tetrazolium salt WST-8, which is reduced by dehydrogenases in viable cells to produce a formazan dye. The amount of formazan dye generated is directly proportional to the number of living cells, which can be quantified by measuring the absorbance.

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Lab products found in correlation

In vitro angiogenesis assay kit, by Merck Group (1 mentions) Oxiselect ros assay kit, by Cell Biolabs (1 mentions) Phase contrast microscope, by Nikon (1 mentions) Multiskan ascent plate reader, by Thermo Fisher Scientific (1 mentions) Lsr 2, by BD (1 mentions) Prism 5, by GraphPad (1 mentions) Egmtm 2 bulletkittm, by Lonza (1 mentions) Formamide, by Thermo Fisher Scientific (1 mentions) Reduced l glutathione, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium, by Wisent (1 mentions) Tubulin tracker green, by Thermo Fisher Scientific (1 mentions) Tecan200pro plate reader, by Tecan (1 mentions) Infinite m200, by Tecan (1 mentions)

13 protocols using wst 8 cell proliferation assay kit

5-FU and FUDR Treatment Optimization

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For drug treatment, 5-FU (Sigma, #F6627) was dissolved in DMSO and FUDR (Sigma, #F0503) was dissolved in water as 100mM stock solutions. The stocks were added to the growth media to the desired final concentrations with or without RNAi and cell viability was tested after 3 days. Cell viability was determined using the WST-8 Cell Proliferation Assay Kit (Cayman Chemical, #10010199) following the manufacturer’s instructions. IC₅₀ was calculated using Prism GraphPad. Each experiment contained 3 replicates and two independent experiments were carried out.

Jia F., Chi C, & Han M. (2020). Regulation of Nucleotide Metabolism and Germline Proliferation in Response to Nucleotide Imbalance and Genotoxic Stresses by EndoU Nuclease. Cell reports, 30(6), 1848-1861.e5.

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HUVEC Proliferation and Migration

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Human umbilical vein endothelial cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates, treated with 5 mM VPA or diluent and cell proliferation was evaluated post-24 and 48 h treatment using WST-8 Cell Proliferation Assay Kit (Cayman Chemicals) according to the manufacturer’s instructions. Migratory capacity of HUVECs were evaluated using Cytoselect^TM 24-well Cell Migration and Invasion Assay kit according to the manufacturer’s protocol (Cell Biolabs, Inc).

Murugavel S., Bugyei-Twum A., Matkar P.N., Al-Mubarak H., Chen H.H., Adam M., Jain S., Narang T., Abdin R.M., Qadura M., Connelly K.A., Leong-Poi H, & Singh K.K. (2018). Valproic Acid Induces Endothelial-to-Mesenchymal Transition-Like Phenotypic Switching. Frontiers in Pharmacology, 9, 737.

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Regulation of HUVEC Proliferation and Angiogenesis by oxLDL

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HUVECs were seeded at a density of 1–1.5 × 10⁴ cells/well in 96‐well plates, transfected with siBRCA2 or scrambled control and then treated with oxLDL, and cell proliferation was evaluated using the WST‐8 Cell Proliferation Assay Kit (Cayman Chemicals) according to the manufacturer's instructions. The angiogenic properties of HUVECs were examined using the In Vitro Angiogenesis Assay Kit (Chemicon): HUVECs were seeded onto ECMatrix Gel–coated 96‐well plates (Millipore, Billerica, Mass) at a density of 7–9 × 10³/well and the extent of their angiogenesis was determined with the aid of a phase‐contrast microscope (Nikon). Each experiment was performed thrice in triplicates. HUVECs underwent silencing before they were treated for 24 hr with oxLDL. ROS were determined using the OxiSelect ROS Assay Kit (Cell Biolabs, Inc).

Singh S., Nguyen H., Michels D., Bazinet H., Matkar P.N., Liu Z., Esene L., Adam M., Bugyei‐Twum A., Mebrahtu E., Joseph J., Ehsan M., Chen H.H., Qadura M, & Singh K.K. (2020). BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL‐induced DNA damage and endothelial apoptosis. Physiological Reports, 8(13), e14481.

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Cell Cycle Analysis and Growth Inhibition

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For cell cycle analysis, cells were serum-starved for 24 hr and were returned to serum-containing media (10% FBS) with the solvent or compounds for various periods as indicated in the figures. After trypsinization, cells were fixed and stained with propidium iodide (PI). PI-labeled cells were counted using a fluorescence activated cell sorter BD LSR II (BD Biosciences, San Jose, CA) and cell cycle distribution was calculated with the Flow Jo software.
For cell growth inhibition, cells were seeded at a density of 8,000 cells per well in 96-well culture plates and allowed to grow for 24 hrs followed by treatment with increasing concentrations of the testing compounds for 3 days. WST-8 mixture from the WST-8 Cell Proliferation Assay Kit (Cayman chemical) was added to each well and incubated for 2 hrs at 37°C. Optical density (OD) values were measured at 450 nm on a Multiskan Ascent plate reader (Thermo Labsystems, Beverly, MA). The curve fit was conducted by applying compound concentration (log value) vs. normalized inhibitory response (percentage value) with variable slope and EC₅₀ values were calculated using Graphpad Prism 5.0 software.

He C., Duan S., Dong L., Wang Y., Hu Q., Liu C., Forrest M.L., Holzbeierlein J.M., Han S, & Li B. (2017). Characterization of a novel p110β-specific inhibitor BL140 that overcomes MDV3100-resistance in castration-resistant prostate cancer cells. The Prostate, 77(11), 1187-1198.

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Cytotoxicity Assay for HCC Cells

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HCC cells were plated in 96-well microtiter plates, with 3000 cells per well, and incubated in 10% FBS-containing cell culture medium overnight at 37 °C. Cells were then treated with the vehicle or various concentrations of the compound in medium for 72 h. Viable cells were quantified using the WST-8 cell proliferation assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s recommended protocol. Results were determined through measurement of the absorbance at 490 nm using a Perkin Elmer Wallac 1420 VICTOR2 microplate reader (Shelton, CT, USA). The IC₅₀ value was defined as the compound concentration that induced a 50% reduction in cell viability in comparison with the DMSO-treated (vehicle) control; this was calculated using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA).

Lai Y.L., Wang K.H., Hsieh H.P, & Yen W.C. (2022). Novel FLT3/AURK multikinase inhibitor is efficacious against sorafenib-refractory and sorafenib-resistant hepatocellular carcinoma. Journal of Biomedical Science, 29, 5.

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Investigating Cell Proliferation and Migration

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HCAECs were seeded at a density of 1–1.5×10⁴ cells/well in 96-well plates, transfected with siIft88 or scrambled control. Cell proliferation was evaluated 24 hrs post-transfection using WST-8 Cell Proliferation Assay Kit (Cayman Chemicals) according to the manufacturer’s guidelines. Following 24 hrs of transfection with either siIft88 or scrambled control, HPAECs were re-seeded in a density of 2×10⁵ cells/well in the upper chamber of the Cyto-Select 24-well Cell Migration Fluorometric Assay system (Cell Biolabs, Inc). Cell migration was evaluated after 12-hrs of incubation according to the manufacturer’s instruction.

Singh S., Adam M., Matkar P.N., Bugyei-Twum A., Desjardins J.F., Chen H.H., Nguyen H., Bazinet H., Michels D., Liu Z., Mebrahtu E., Esene L., Joseph J., Ehsan M., Qadura M., Connelly K.A., Leong-Poi H, & Singh K.K. (2020). Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis. Scientific Reports, 10, 4466.

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HUVEC Proliferation Assay with siRNA

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HUVECs were first cultured in endothelial cell growth medium-2 (EGM^TM-2 Bulletkit^TM; Lonza) supplemented with growth factors, serum and antibiotics at 37°C in humidified 5% CO₂, and seeded at a density of 1–2×10⁴ cells/well in 96-well plates, transfected with sieNOS (5 nmol) or scrambled control (5 nmol), and then cell proliferation was evaluated 24, 48 and 72 hrs post-transfection using WST-8 Cell Proliferation Assay Kit (Cayman Chemicals) as described [15 (link)]. HUVECs were transfected in a 6-well plate (1.5–2×10⁵ cells/well) with either sieNOS or scrambled control and cells were counted using CytoSmart cell counter 24, 48 and 72 hrs post-transfection. Cells were counted in triplicates for each biological replicate and average cell number was determined for each sample.

Bu S., Nguyen H.C., Nikfarjam S., Michels D.C., Rasheed B., Maheshkumar S., Singh S, & Singh K.K. (2022). Endothelial cell-specific loss of eNOS differentially affects endothelial function. PLoS ONE, 17(9), e0274487.

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Cellular Proliferation Assay Protocol

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Formamide (≥99.5%) and reduced l-glutathione (≥98.0%) were purchased from Thermo Scientific. Phosphate buffer solution (PBS, 1×) and Dulbecco's modified Eagle medium (DMEM) were purchased from Wisent. HyClone™ Calf Serum and Tubulin Tracker Green™ were purchased from Thermo Scientific. The WST-8 Cell Proliferation Assay Kit was purchased from Cayman Chemical. All reagents were used without further modification or purification.

Macairan J.R., Jaunky D.B., Piekny A, & Naccache R. (2018). Intracellular ratiometric temperature sensing using fluorescent carbon dots. Nanoscale Advances, 1(1), 105-113.

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Cell Viability Assay for C75 and Colchicine

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Assays were performed to determine the viability of cells after treatment with C75 and/or colchicine. The concentration of colchicine or C75 used in the combination experiments was the highest concentration that caused little to no toxicity based on dose–response curves. HFF-1, HeLa, A549 and HCT 116 cells were plated with 4,000 cells/well in 96-well dishes and left overnight to adhere. Drug dilutions were prepared and added to the cells using an acoustic liquid handler (LabCyte ECHO 550). After 3 population doubling times, cells were assessed for viability using the WST-8 cell proliferation assay kit (Cayman Chemicals). Absorbance readings at 450 nm were collected using the TECAN 200 PRO plate reader. Values for each replicate were adjusted to the controls and plotted using Graphpad Prism v.9.2.0 to generate graphs and IC₅₀ values. All assays were performed in triplicate for each treatment. For the combination assays, C75 or colchicine were repeated alongside the combination treatments to ensure accurate comparison.

Jaunky D.B., Larocque K., Husser M.C., Liu J.T., Forgione P, & Piekny A. (2021). Characterization of a recently synthesized microtubule-targeting compound that disrupts mitotic spindle poles in human cells. Scientific Reports, 11, 23665.

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5-FU and FUDR Treatment Optimization

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