Celltiter 96 aqueous

Manufactured by Promega

Sourced in United States, France, United Kingdom, Canada

CellTiter 96 AQueous is a colorimetric assay for determining the number of viable cells in proliferation or cytotoxicity assays. It utilizes a novel tetrazolium compound that is bioreduced by metabolically active cells into a colored formazan product that is soluble in tissue culture medium.

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Lab products found in correlation

Microplate reader, by Agilent Technologies (3 mentions) 96 well plate, by Corning (3 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Deferoxamine mesylate, by Merck Group (2 mentions) Ferrostatin 1, by Selleck Chemicals (2 mentions) Erastin, by Selleck Chemicals (2 mentions) Elisa plate reader, by Tecan (2 mentions) Prism 8, by GraphPad (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) M200 microplate reader, by Tecan (2 mentions) Thermomax, by Molecular Devices (2 mentions) Sdf 1, by Merck Group (2 mentions) T75 flask, by Greiner (1 mentions) Fluostar omega plate reader, by BMG Labtech (1 mentions) Live cell analysis system, by Sartorius (1 mentions) Cell proliferation elisa brdu assay, by Roche (1 mentions) Incucyte live cell system, by Sartorius (1 mentions) Boceprevir, by Merck Group (1 mentions) Recombinant human rage fc chimera, by R&D Systems (1 mentions) Recombinant human hmgb1, by R&D Systems (1 mentions)

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88 protocols using celltiter 96 aqueous

Cell Viability Assay Protocol

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To evaluated the cell viability, CellTiter 96 AQueous (Promega) was added to PC12 cells or primary cortical neurons at 1/10 volume of the medium. Cells were incubated at 37°C for 4 h. Two hundred microliters of mixed medium was transferred into 96-well ELISA plate. The absorbance was measured at 490 nm using a Microplate reader (BIO-RAD). Cell viability was presented as the percentage of absorbance obtained in control cells.

Ye X., Luo H., Chen Y., Wu Q., Xiong Y., Zhu J., Diao Y., Wu Z., Miao J, & Wan J. (2015). MicroRNAs 99b-5p/100-5p Regulated by Endoplasmic Reticulum Stress are Involved in Abeta-Induced Pathologies. Frontiers in Aging Neuroscience, 7, 210.

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Cytotoxicity Screening of Test Solutions

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Test solutions were analysed for cytotoxicity using the Cell Titer 96 AQueous assay method (Promega, Southampton, UK) and were performed in a 96 well plate (96WP) with a known concentration of Vero cells in each well. A density of 2.5 x 10³ cells per well were used for each plate prepared, by the trypsinisation of 80% confluent Vero cells in a T75 flask (Greiner Bio-One, Stonehouse, UK) and reconstituted in fresh DMEM Cells were seeded into a 96WP and incubated for 24 h at 37°C under 5% CO₂. The medium was removed by aspiration, the cells washed three times with phosphate-buffered saline then immediately replaced with the dilutions of test materials. Cells were incubated for 6, 24, 48 and 72 h. Cell Titer 96 AQueous solution was used to detect cell viability and was added to each well and the plates incubated for 1 h at 37°C under 5% CO₂. Optical density was then determined at 492 nm, with the blank subtracted from each sample reading and the density mean for the control cells arbitrarily assigned a value of 100% (n = 3).

Houston D.M., Bugert J.J., Denyer S.P, & Heard C.M. (2017). Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV. PLoS ONE, 12(6), e0179291.

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MTS-based Cell Proliferation Assay

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A cell proliferation assay (CellTiter 96 AQueous; Promega) was used in accordance with the manufacturer’s instructions to assess FHL124 cell viability. This assay is a colorimetric method for determining the number of viable cells in proliferation. The assay is based on the cellular conversion of a tetrazolium salt (MTS) into a formazan product. The resultant absorbance is directly proportional to the number of viable cells in culture. Absorbance was measured at 490 nm with a spectrophotometric plate reader (FLUOstar Omega plate reader; BMG Labtech).

Liu H., Smith A.J., Ball S.S., Bao Y., Bowater R.P., Wang N, & Michael Wormstone I. (2017). Sulforaphane promotes ER stress, autophagy, and cell death: implications for cataract surgery. Journal of Molecular Medicine (Berlin, Germany), 95(5), 553-564.

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Antiestrogen Response in Breast Cancer Cell Lines

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MCF-7 and other selected BC cells were seeded in 96-well plates at 3–5 × 10⁵ cells/well in complete medium. After 24 hours, medium was switched to estrogen-free conditions as described above. After 48 hrs, cells were treated with indicated concentrations of antiestrogens for 72 hrs with or without estradiol-17β (E2). Cell number and viability were determined by either cell counts or by colorimetric assays using the CellTiter 96 Aqueous (Promega) assay or the cell proliferation ELISA BrdU assay (Roche) as per manufacturer’s instructions. Treatments were done in quadruplicate, and experiments were repeated at least three times. In selected experiments using the Incucyte™ Live Cell System (Essen Bioscience) as per the manufacturer’s instructions, the proliferation of 4T1 cells maintained in a tissue culture incubator was monitored by using the NucLight Rapid Red Reagent for cell labeling in 6-well plates. Images for cell confluence were obtained every 4–6 hrs; as cells proliferate, the confluence increases, and confluence is therefore a surrogate for proliferation. Images were analyzed using the Live-Cell Analysis System (Essen Bioscience).

Márquez-Garbán D.C., Deng G., Comin-Anduix B., Garcia A.J., Xing Y., Chen H.W., Cheung-Lau G., Hamilton N., Jung M.E, & Pietras R.J. (2019). Antiestrogens in combination with immune checkpoint inhibitors in breast cancer immunotherapy. The Journal of steroid biochemistry and molecular biology, 193, 105415.

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Cytotoxicity Assessment via MTT Assay

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The MTT assay was used to measure compounds cytotoxicity by applying 20/well of the MTT solution (CellTiter 96® AQueous, Promega) and incubating the cells for 4 h at 37 °C and 5% CO₂. The absorbance was measured at 490 nm using a 96-well plate reader.

Rothan H.A, & Teoh T.C. (2021). Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro). Molecular Biotechnology, 63(3), 240-248.

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Cytocompatibility Evaluation of Nanoparticles

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To investigate cytocompatibility, a cell viability assay using an MTS assay (CellTiter 96® AQueous, Promega, Madison, WI) was performed. Polarized U937 cells (5 × 10³ per well) were seeded in 96-well plates. After 24 h, cells were treated with 0–200 μg/mL of F127-TA core nanoparticles or MDNPs in medium for 24 h. Then, cells were incubated with 20 µL MTS reagent in each well and allowed to develop color for 2 h at 37 °C. The absorbance of developed color is proportional to cell growth, and this was measured at 490 nm in a microplate reader (BioTeK Cytation 3, Winooski, VT, USA). The effect of nanoparticles on the cell growth of U937 was measured as the percentage of cell viability, where control cells (no treatment group) was considered 100% viable.

Hatami E., Mu Y., Shields D.N., Chauhan S.C., Kumar S., Cory T.J, & Yallapu M.M. (2019). Mannose-decorated hybrid nanoparticles for enhanced macrophage targeting. Biochemistry and Biophysics Reports, 17, 197-207.

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Screening for SARS-CoV-2 3CLpro Inhibitors

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A small in-house library was screened by treating cells with the compounds prepared in DMSO at 10 µM concentration for 48 h. We used boceprevir (Cas # 394730600, Sigma) as a positive control that showed activity against SARS-COV-2 3CLpro [39 (link)]. The experiments were run on three occasions to identify the assay's hit rate, the reproducibility of the assay, and the false negative and false positive hit rates in the assay. At the endpoint of the split-GFP-3CLpro assay, we measure the compound cytotoxicity by applying 20/well of the MTT solution (CellTiter 96® AQueous, Promega) and incubating the cells for 4 h at 37 °C and 5% CO₂. The absorbance was measured at 490 nm using a 96-well plate reader.

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Virus Growth Kinetics and Cytotoxicity Assay

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In order to determine the growth kinetics of the virus, cancer cells were seeded in 6 well plates at the density of 300000/well in 2 mL cell culture medium. Next day, cells were infected with the virus at multiplicity of infection (MOI) 0.03. Cell lysates were collected at 24-, 48- and 72-h post-infection and virus titer in the lysates were determined by standard plaque assay on CV1 cells as described previously.²¹To determine the cytotoxic potential of the virus, 3000 cells were seeded per well of 96-well culture plates in 100 uL cell culture medium. The following day, cells were either mock-infected or infected by CF33-hNIS-ΔF14.5 at MOIs 0.01, 0.1 or 1. After infection, plates were incubated for 72 h. Next, CellTiter96®AQueous (Promega) reagent was added to the cells and 1 h later absorbance was measured at 490 nm using a plate reader (Tecan Spark) as per the manufacturer’s instruction. Survival was calculated relative to mock-infected wells.

Chaurasiya S., Yang A., Kang S., Lu J., Kim S.I., Park A.K., Sivanandam V., Zhang Z., Woo Y., Warner S.G, & Fong Y. (2020). Oncolytic poxvirus CF33-hNIS-ΔF14.5 favorably modulates tumor immune microenvironment and works synergistically with anti-PD-L1 antibody in a triple-negative breast cancer model. Oncoimmunology, 9(1), 1729300.

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Evaluating HMGB1-RAGE Signaling in Cholangiocytes

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Human immortalized nonmalignant cholangiocyte H69 cells were cultured in 96-well plates (5,000 cells/well) (16 (link)). After 24 hours, cells were cultured with DMEM/F-12 medium/0.5% serum ± pre-treatment with 5 ug/mL recombinant human RAGE-Fc chimera (R&D, Minneapolis, MN) for 1 hour ± 100 ng/mL recombinant human HMGB1 (R&D) for 48 hours. Cellular proliferation was quantified with a colorimetric assay (CellTiter 96Aqueous; Promega). RNA was isolated from H69 cells in different wells using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and subjected to real-time PCR with the Brilliant III SYBR Green QPCR Master Mix Gene Expression Assay Kit and the Mx3005p system (Stratagene), normalized with GAPDH. Primer sequences are listed in Supplemental Table 8.

Luo Z., Jegga A.G, & Bezerra J.A. (2018). Gene-disease associations identify a connectome with shared molecular pathways in human cholangiopathies. Hepatology (Baltimore, Md.), 67(2), 676-689.

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Modulating GBM TICs Proliferation by miR-100

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GBM TICs were grown as spheres, disseminated into single cells, then inoculated in 96-well plates at a density of 20,000 cells/well and temperature of 37 oC. After 1 day of growth, the cells were transfected with pre-miR-100 or with a control miR. Following 2 days of growth in culture medium containing 4.5 g glucose DMEM/F12, 20 pg EGF and 20 pg bFGF, cell numbers were quantified using an MTS assay (CellTiter 96 Aqueous, Promega, USA) as per the manufacturer’s instructions.

Alrfaei B.M., Clark P., Vemuganti R, & Kuo J.S. (2020). MicroRNA miR-100 Decreases Glioblastoma Growth by Targeting SMARCA5 and ErbB3 in Tumor-Initiating Cells. Technology in Cancer Research & Treatment, 19, 1533033820960748.

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