Colorimetric xtt assay

Manufactured by Roche

Sourced in Germany

The Colorimetric XTT assay is a laboratory equipment product designed to measure cell viability and proliferation. It utilizes a tetrazolium salt, XTT, which is reduced by metabolically active cells to produce a colored formazan product. The intensity of the color change is directly proportional to the number of living cells, allowing for quantitative assessment of cell health and growth.

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Lab products found in correlation

Xcelligence system, by Roche (1 mentions) Stat fax 2100, by Awareness Technology (1 mentions) Astemizole, by Merck Group (1 mentions) Gefitinib, by AstraZeneca (1 mentions)

Spelling variants of this product

XTT assay (92 citations) Colorimetric assay (27 citations) Colorimetric XTT Assay Kit (2 citations)

8 protocols using colorimetric xtt assay

Synergistic Anti-Tumor Effects of Gemcitabine and Entinostat

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3×10³- 5×10³ cells were seeded in 96-well microtitre plates and allowed to adhere overnight before adding indicated amounts of gemcitabine and/or entinostat. After 3 days of treatment, cell viability was measured using the XTT colorimetric assay (Roche and Promega) according to the manufacturer's protocol. Viability was plotted as percentage viability compared to untreated control. Results were calculated from at least two independent experiments with triplicates each time, and all data are presented as means +/− SE. Student's t-tests for paired data were employed and considered as significant as follows: *P < 0.05; **P < 0.01; and ***P < 0.001.
For each of the experiments, the extent and direction of antitumour interactions between gemcitabine and entinostat were determined by calculating combination index (CI) using the median dose–effect method of Chau and Talalay, and CompuSyn software. CI <1, CI = 1, CI >1 indicates synergistic, additive or antagonistic effects respectively [23 (link)–25 ].

Ma Y.T., Leonard S.M., Gordon N., Anderton J., James C., Huen D., Woodman C.B, & Palmer D.H. (2017). Use of a genome-wide haploid genetic screen to identify treatment predicting factors: a proof-of-principle study in pancreatic cancer. Oncotarget, 8(38), 63635-63645.

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Evaluating Cell Proliferation and Viability

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Cell proliferation and viability was assessed using an XTT colorimetric assay (Roche Applied Science). Cells were seeded on 96-well plates at a density of 4,000 cells/well and let to attach for 24 hours. The cells were then treated with AEE788 (0–20 μM) as a single agent or in combination with celecoxib (10 μM) in the presence or absence of EGF (100 ng/mL). Controls cells were treated with the same concentration of the DMSO vehicle. After treatment at indicated times and doses, the XTT assay was performed following the protocol supplied by the manufacturer using a microplate absorbance reader.

Valverde A., Peñarando J., Cañas A., López-Sánchez L.M., Conde F., Hernández V., Peralbo E., López-Pedrera C., de la Haba-Rodríguez J., Aranda E, & Rodríguez-Ariza A. (2015). Simultaneous Inhibition of EGFR/VEGFR and Cyclooxygenase-2 Targets Stemness-Related Pathways in Colorectal Cancer Cells. PLoS ONE, 10(6), e0131363.

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Colorimetric XTT Cell Proliferation Assay

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The colorimetric XTT assay (Roche) was used according to the manufacturer’s instructions for quantification of cell proliferation [30 (link)].

Mahli A., Seitz T., Beckröge T., Freese K., Thasler W.E., Benkert M., Dietrich P., Weiskirchen R., Bosserhoff A, & Hellerbrand C. (2019). Bone Morphogenetic Protein-8B Expression is Induced in Steatotic Hepatocytes and Promotes Hepatic Steatosis and Inflammation In Vitro. Cells, 8(5), 457.

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Measuring Cell Proliferation and Mitochondrial Activity

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Cell proliferation was measured using the xCELLigence system (Roche) according to the manufacturer's instructions. In addition, cell proliferation was determined by cell counting. Here, 1 × 10⁶ cells were seeded into T75 flasks. After 48h, cells were trypsinized and microscopically counted. Mitochondrial activity was measured applying a colorimetric XTT assay (Roche) [51 (link)].

Koch A., Lang S.A., Wild P.J., Gantner S., Mahli A., Spanier G., Berneburg M., Müller M., Bosserhoff A.K, & Hellerbrand C. (2015). Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells. Oncotarget, 6(32), 32748-32760.

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Cell Viability Assay with Antivirals

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Cell viability following treatment with DHA, PA, 3TC, or AZT was assessed using a colorimetric XTT assay (Roche Basel, Switzerland, 11465015001). To initiate the assay, 4 × 10⁴ cells were seeded into wells in a 96-well plate. After a 24 h incubation period, allowing for cellular attachment, the culture medium was replaced, and the specified concentrations of PA, DHA, 3TC, or AZT were introduced to the cells. The incubation was carried out for 24 h at 37 °C, under conditions of 5% CO₂ and 95% humidity. A freshly prepared XTT mixture was employed for the assay, created by blending the XTT labeling reagent with the electron coupling reagent at a ratio of 50:1. Subsequently, 50 µL of the XTT mixture was added to the cells, and the cells were further incubated for 18 h at 37 °C, maintaining an environment of 5% CO₂ and 95% humidity, adhering to the manufacturer’s protocol. Following this incubation period, the absorbance of the samples was determined at 450 nm, with reference measurements taken at wavelengths exceeding 650 nm. This analysis was conducted utilizing a Synergie multi-well scanning spectrophotometer (STAT FAX 2100, Awareness Technology, Inc., Palm City, FL, USA).

Lipke K., Kubis-Kubiak A, & Piwowar A. (2023). The Influence of Nucleoside Reverse Transcriptase Inhibitors on Mitochondrial Activity, Lipid Content, and Fatty-Acid-Binding Protein Levels in Microglial HMC3 Cells. Pharmaceuticals, 16(12), 1661.

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Quantifying Hepatocellular Mitochondrial Activity

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For quantification of hepatocellular mitochondrial activity, the colorimetric XTT assay (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions.

Mahli A., Thasler W.E., Patsenker E., Müller S., Stickel F., Müller M., Seitz H.K., Cederbaum A.I, & Hellerbrand C. (2015). Identification of cytochrome CYP2E1 as critical mediator of synergistic effects of alcohol and cellular lipid accumulation in hepatocytes in vitro. Oncotarget, 6(39), 41464-41478.

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Quantifying Hepatocellular Mitochondrial Activity

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For quantification of hepatocellular mitochondrial activity, the colorimetric XTT assay (Roche Diagnostics, Mannheim, Germany) was used according to the manufacturer's instructions.

Sommer J., Mahli A., Freese K., Schiergens T.S., Kuecuekoktay F.S., Teufel A., Thasler W.E., Müller M., Bosserhoff A.K, & Hellerbrand C. (2016). Analysis of molecular mechanisms of 5-fluorouracil-induced steatosis and inflammation in vitro and in mice. Oncotarget, 8(8), 13059-13072.

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Assessing Synergistic Effects of Astemizole and Gefitinib

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Cells were seeded in 96-well plates at a density of 1000 cells per well. After 24 h, cells were incubated in the presence of different concentrations of astemizole (Sigma, St. Louis, MO, USA), gefitinib (kindly provided by AstraZeneca, Mexico City, Mexico), or the vehicle alone (dimethyl sulfoxide, DMSO) during 6 days. Cell proliferation was studied using the colorimetric XTT assay (Roche Applied Sciences, IN, USA) following the manufacturer's instructions. Absorbance was determined at 492 nm in a Multiskan spectrophotometer (Labsystems Inc., Canada). Furthermore, cell density was determined using the protein-binding dye SRB 30 . The concentrations that caused 20% and 50% cell proliferation inhibition (inhibitory concentrations [IC] 20 and IC 50 , respectively) were calculated by non-linear regression analysis using sigmoidal fitting from the sigmoidal dose-response curve by means of the scientific graphing software Origin (OriginLab Corporation, Northampton, MA). Subsequently, using the IC 20 and IC 50 of astemizole and gefitinib, we studied the effect of the drug combination to determine a possible synergism on cell proliferation. Then, combination index (CI) values were determined applying CI equation 31 . For this analysis, an additive effect, synergism, or antagonism were defined as CI values = 1.0, < 1.0, and > 1.0, respectively, as previously reported 31 .

García-Quiroz J., González-González M.E., Díaz L., Ordaz-Rosado D., Segovia-Mendoza M., Prado-García H., Larrea F, & García-Becerra R. (2019). ASTEMIZOLE, AN INHIBITOR OF ETHER-À-GO-GO-1 POTASSIUM CHANNEL, INCREASES THE ACTIVITY OF THE TYROSINE KINASE INHIBITOR GEFITINIB IN BREAST CANCER CELLS. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion, 71(3).

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