Celltiter 96 reagent

Manufactured by Promega

Sourced in United States

The CellTiter 96 reagent is a colorimetric assay that measures the number of viable cells in a sample. The assay utilizes a tetrazolium compound that is reduced by metabolically active cells, producing a colored formazan product. The amount of formazan produced is directly proportional to the number of viable cells, which can be measured using a spectrophotometer.

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Lab products found in correlation

Prestoblue, by Thermo Fisher Scientific (2 mentions) Alamar blue, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions) 6 well plate, by Corning (1 mentions) Ula plate, by Corning (1 mentions) Flexstation 3, by Molecular Devices (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

MTS assay (CellTiter 96 Aqueous One Solution reagent) CellTiter 96-well assay reagent CellTiter 96® Aqueous One Solution Non-Radioactive (MTS) Cell Proliferation Assay Reagent CellTiter 96® AQ One Solution Reagent CellTiter 96 AQueous One Solution Proliferation Assay reagent CellTiter 96 AQueous One Solution MTS Reagent CellTiter 96 AQueous MTS Reagent MTS assay Kit (CellTiter 96 Aqueous One Solution reagent) CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) reagent CellTiter 96® AQueous reagent

13 protocols using celltiter 96 reagent

Adenovirus Cytotoxicity and Viral Yield Analysis

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For AdV cytotoxicity assay, at 6d post-infection plates were stained with tetrazolium-based MTS (CellTiter96 Reagent, Promega) and the microtiter plates were read spectrophotometrically at 490/650nm (Molecular Devices).
Prior to assessing cell viability using MTS, supernatants were collected from plates and titrated to determine virus yield. Serial dilutions of supernatants were incubated on HeLa cells for 3 days. Following incubation, wells were visually scored for infection and TCID₅₀, EC₅₀, CC₅₀ and SI were calculated for each sample.

Widman D.G., Gornisiewicz S., Shacham S, & Tamir S. (2018). In vitro toxicity and efficacy of verdinexor, an exportin 1 inhibitor, on opportunistic viruses affecting immunocompromised individuals. PLoS ONE, 13(10), e0200043.

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Cytotoxicity Assay for Compounds

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The cytotoxic effect of the compounds was determined using the identical cell culture procedure to that described above for viral replication or cell-cell fusion, but measuring cell viability, using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, CellTiter 96 reagent; Promega; used for MAGI assays) or using a resazurin cell viability reagent (Alamar Blue or Presto Blue, Life Technologies; used for cell-cell fusion assays) following the manufacturers’ protocols.

Sofiyev V., Kaur H., Snyder B.A., Hogan P.A., Ptak R.G., Hwang P, & Gochin M. (2016). Enhanced potency of bivalent small molecule gp41 inhibitors. Bioorganic & medicinal chemistry, 25(1), 408-420.

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Colorimetric Assays for Cell Proliferation

Cited 1 time

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For CCK-8 assay, CRC cells were seeded into 96-well plates at a density of 1 × 10³ per well. Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added into each well at indicated time points. After incubation at 37 °C for 2 h, the absorbance was measured using an automatic microplate at 450 nm.
For EdU (5-ethynyl-2′-deoxyuridine) assay, exponentially growing cells plated on 6-well plates (Corning) were labelled as described [21 (link)]. Then EdU-labeled or unlabeled LoVo and HCT116 cells were cultured into a 96-well plate at 2 × 10³ cells/well in McCoy’s 5A medium added with 10% FBS at 37 °C. At 0, 24, 48, 72, and 96 h, 10 μl of Cell Titer-96 reagent (Promega Inc., Madison, WI) was supplemented to each well. After 1 h of further incubation at 37 °C, the cells were scanned at the wavelength of 490 nm in a plate reader (Molecular Devices Corp., Sunnyvale, CA).

Li J., He M., Xu W, & Huang S. (2019). LINC01354 interacting with hnRNP-D contributes to the proliferation and metastasis in colorectal cancer through activating Wnt/β-catenin signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 161.

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Antiviral Activity Measurement of R430

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Antiviral activity of R430 was determined by measuring the virus associated extracellular HBV DNA in HepG2.2.2.15 cells using real-time qPCR (TaqMan) as published^{34 (link)}. Briefly, HepG2 2.2.15 cells were plated in 96-well plates at sub-confluent levels, followed by treatment in triplicates with serially diluted compounds next day and incubated at 37 °C with 5% CO₂. At three days post-treatment, cell culture media was replenished with fresh compounds. Six days after the experiment initiation, cell culture supernatant was treated with pronase and used for the measurement of the virus associated DNA using real time qPCR, according to the standard protocol. Extracellular HBV DNA copy numbers were normalized to untreated controls to determine EC50 and EC90. Antiviral compound, Lamivudine (3TC), was used as a positive control in each assay. Cytotoxicity was measured by CellTiter® 96 Reagent, (Promega) uptake assay to determine CC50. Selectivity index 50% (SI50) were calculated by the ratio of CC50/EC50 and CC50/EC90.

D’Aiuto L., McNulty J., Hartline C., Demers M., Kalkeri R., Wood J., McClain L., Chattopadhyay A., Zhi Y., Naciri J., Smith A., Yolken R., Chowdari K., Zepeda-Velazquez C., Dokuburra C.B., Marques E., Ptak R., Kinchington P., Watkins S., Prichard M., Bloom D, & Nimgaonkar V. (2018). R430: A potent inhibitor of DNA and RNA viruses. Scientific Reports, 8, 16662.

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Cytotoxicity Assay for Compound Screening

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The cytotoxic
effect of the compounds
was determined using the cell culture procedure identical to that
described above for viral replication or cell–cell fusion but
measuring cell viability using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, CellTiter 96 reagent; Promega; used
for MAGI and PBMC assays) or using a resazurin cell viability reagent
(Alamar Blue or Presto Blue, Life Technologies; used for cell–cell
fusion assays) following the manufacturers’ protocols.

Zhou G., Sofiyev V., Kaur H., Snyder B.A., Mankowski M.K., Hogan P.A., Ptak R.G, & Gochin M. (2014). Structure–Activity Relationship Studies of Indole-Based Compounds as Small Molecule HIV-1 Fusion Inhibitors Targeting Glycoprotein 41. Journal of Medicinal Chemistry, 57(12), 5270-5281.

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Zika Virus Cytoprotection Assay

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Example 11

The Zika virus cytoprotection assay uses Vero cells and strain PRVABC59. Briefly, virus and cells are mixed in the presence of test compound and incubated for 5 days. The virus is pre-titered such that control wells exhibit 85 to 95% loss of cell viability due to virus replication. Therefore, antiviral effect is assessed as a function of cytoprotection. Cytoprotection and compound cytotoxicity are assessed by MTS (CellTiter®96 Reagent, Promega, Madison WI) reduction. The % reduction in viral cytopathic effects (CPE) is determined and reported; EC50 (concentration inhibiting virus-induced cytopathic effects by 50%), CC50 (concentration resulting in 50% cell death) and a calculated SI (selectivity index=CC50/EC50) are provided along with a graphical representation of the antiviral activity and compound cytotoxicity when compounds are tested in dose-response. Each assay includes Interferon-β as a positive control.

US11801238B2. Anti-viral activity of VPS34 inhibitors (2023-10-31). Deciphera Pharmaceuticals, LLC [US]. Inventors: Daniel L. Flynn [US].

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Antiviral Evaluation of Remdesivir

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Antiviral assessments of RDV for enteroviruses, rhinoviruses, and influenza viruses were conducted in RD, H1 HeLa, and MDCK cells, respectively, using cytopathic effect (CPE) assays. Virus and cells were mixed in the presence of test compound and incubated for the indicated duration. The virus was pre-titered such that control wells exhibited 85 to 95% loss of cell viability due to virus replication. Therefore, antiviral effect or cytoprotection was observed when compounds prevented virus replication. Cytoprotection and compound cytotoxicity was assessed by MTS (CellTiter®96 Reagent, Promega) dye reduction. The % reduction in viral cytopathic effects (CPE) was determined and used to calculate EC₅₀ (concentration inhibiting virus replication by 50%) and CC₅₀ (concentration resulting in 50% cell death).

Radoshitzky S.R., Iversen P., Lu X., Zou J., Kaptein S.J., Stuthman K.S., Van Tongeren S.A., Steffens J., Gong R., Truong H., Sapre A.A., Yang H., Xie X., Chia J.J., Song Z.J., Leventhal S.M., Chan J., Shornikov A., Zhang X., Cowfer D., Yu H., Warren T., Cihlar T., Porter D.P., Neyts J., Shi P.Y., Wells J., Bilello J.P, & Feng J.Y. (2023). Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro. Scientific Reports, 13, 3131.

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MTS Assay for Cell Viability

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Cell viability was determined using 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay as previously described [14] (link). Briefly, 2000 cells per well were plated in full growth media with or without doxycycline (2 μg/ml) for 0, 24, 48, and 72 hours, respectively. At each time point, the assay was terminated and relative cell growth was determined using the CellTiter 96 reagent (Promega, Madison, WI), as recommended in the manufacturer's protocol. All experiments were set up in triplicates, and the results were normalized to the mean cell viability at 0 hours.

Bisht S., Nolting J., Schütte U., Haarmann J., Jain P., Shah D., Brossart P., Flaherty P, & Feldmann G. (2015). Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread—Identification of a Promising Novel therapeutic target. Translational Oncology, 8(4), 295-307.

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Niemann–Pick C cell viability assay

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Cell viability was assessed using Promega CellTiter 96 Aqueous One Solution cell proliferation colometric assay (G3580). Briefly, Niemann–Pick C cells were cultured in 96-well plates at 10,000 cells per well for 24 h, washed 3× with PBS, and treated as indicated with compounds diluted in media for 24 h. Cells were washed 3× with PBS and re-suspended in media supplemented with Promega CellTiter 96 reagent (20 μl reagent per 100 μl of media). After 45-min incubation at 37 °C, absorbance was read at 490 nm using a microplate reader. Each treatment was performed in triplicate, and the average absorbance reading of non-treated (Veh) cells was set to 100%. The percent viability was determined by dividing the average absorbance of treated over non-treated cells and multiplying by 100.

Schultz M.L., Fawaz M.V., Azaria R.D., Hollon T.C., Liu E.A., Kunkel T.J., Halseth T.A., Krus K.L., Ming R., Morin E.E., McLoughlin H.S., Bushart D.D., Paulson H.L., Shakkottai V.G., Orringer D.A., Schwendeman A.S, & Lieberman A.P. (2019). Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases. BMC Medicine, 17, 200.

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HIV-1 Cytoprotection Assay with CEM-SS Cells

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The HIV Cytoprotection assay used CEM-SS cells and the IIIB strain of HIV-1. Briefly virus and cells were mixed in the presence of test compound and incubated for 6 days. The virus was pre-titered such that control wells exhibit 70 to 95% loss of cell viability due to virus replication. Therefore, antiviral effect or cytoprotection was observed when compounds prevent virus replication. Each assay plate contained cell control wells (cells only), virus control wells (cells plus virus), compound toxicity control wells (cells plus compound only), compound colorimetric control wells (compound only) as well as experimental wells (compound plus cells plus virus). Cytoprotection and compound cytotoxicity were assessed by MTS (CellTiter® 96 Reagent, Promega, Madison WI) and the EC₅₀ (concentration inhibiting virus replication by 50%), CC₅₀ (concentration resulting in 50% cell death) and a calculated TI (therapeutic index CC₅₀/EC₅₀) were provided. Each assay included AZT as a positive control.

Wu B., Tang J., Wilson D.J., Huber A.D., Casey M.C., Ji J., Kankanala J., Xie J., Sarafianos S.G, & Wang Z. (2016). 3-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides potently inhibit HIV-1 integrase and RNase H. Journal of medicinal chemistry, 59(13), 6136-6148.

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