Xtt assay

Manufactured by Merck Group

Sourced in United States, Poland

The XTT assay is a colorimetric assay used to measure cell viability, proliferation, and cytotoxicity. It is based on the enzymatic conversion of the tetrazolium salt XTT into a colored formazan product, which can be quantified spectrophotometrically. The core function of the XTT assay is to provide a simple, reliable, and quantitative method for assessing cell metabolic activity.

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48 protocols using xtt assay

Evaluating HNSCC Cell Behavior

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Cell proliferation, migration, and invasion assays were performed using HNSCC cells. The XTT assay (Sigma–Aldrich, St. Louis, MO, USA) characterized cell proliferation, a chamber assay using the Corning BioCoat^TM cell culture chamber (Corning, Corning, NY, USA) assessed migration, and Matrigel chamber assays using the Corning BioCoat Matrigel assessed invasion. They were performed with HNSCC cells as described previously [20 (link),21 (link),57 (link),58 (link)]. The reagents used in this study are listed in Supplemental Table S1.

Minemura C., Asai S., Koma A., Kase-Kato I., Tanaka N., Kikkawa N., Kasamatsu A., Yokoe H., Hanazawa T., Uzawa K, & Seki N. (2022). Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma. International Journal of Molecular Sciences, 23(7), 3808.

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Evaluating Antibody Toxicity in Cells

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To evaluate toxicity of the antibodies and pharmacological inhibitors, cells were cultured in the presence or absence of anti-CD81, PD98059 (30μM), SB203580 (10μM) or SP600125 (10μM) for 72h. Then, the cells were stained with propidium iodide (PI, 50μg/ml, BD Bioscience) and analyzed by flow cytometry, using FACSCalibur equipment and CellQuest software. To further confirm the data, the viability of B cells cultured with anti-CD81 were also assessed by XTT assay (Sigma Aldrich), according to the manufacturer’s protocol; and the concentration of lactate dehydrogenase (LDH) in culture supernatant were evaluated, using LDH assay Kit (Doles, Goias, Brasil).

Correa A.R., Berbel A.C., Papa M.P., de Morais A.T., Peçanha L.M, & de Arruda L.B. (2015). Dengue Virus Directly Stimulates Polyclonal B Cell Activation. PLoS ONE, 10(12), e0143391.

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Antimicrobial Efficacy of Peptides against Candida Biofilms

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Example 5

The viability of various C. albicans biofilms (CA494, MYA 2876, CA 2519, CA 526 and ATCC 64124) in the presence of BmKn2, dBmKn2, Kn2-7 or dKn2-7 peptides was measured using the XTT assay (Sigma-Aldrich, St. Louis, Mo., USA) 24 hours after treatment. Results are depicted in FIGS. 3-7, and indicate that dBmKn2 and dKn2-7 caused 100% reduction in biofilm viability in all S fungal strains, and dKn2-7 showed up to a 4-fold higher efficacy against the fungal biofilms compared to the other three peptides. This indicates the D-amino acid forms of the peptides may be more stable against biofilm proteases. We note herein that the ATCC 64124 strain is ketoconazole resistant, and thus the data in FIG. 7 suggest that these peptides have activity against this drug resistant strain of C. albicans.

US11524051B2. Methods of treating fungal infections (2022-12-13). United States of America as represented by the Secretary of the Navy [US]. Inventors: Nancy Millenbaugh [US], Jeremy Wesley Gleaton [US], Dickson Kiprono Kirui [US], Sabrina Snyder [US], Crystal Rock [US].

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Inhibition of HER2-Expressing Cell Lines

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Example 11

The antibodies from the D-series of immunizations were tested for their ability to inhibit growth of the high-HER2 expressing cell lines SKBR3 and BT474. SKBR3 and BT474 cells were plated in ½-area 96-well plates at a density of 120 k/well and 240 k/well, respectively. SKBR3 cells were cultured in 100 μL McCoy's 5A (ATCC) 10% FBS, 1% penicillin-streptomycin and 2 mM L-glutamine. BT474 cells were maintained in 100 μL at 37° C. in 5% CO₂in 50:50 DMEM: F12, 10% FBS, 1% penicillin-streptomycin and 2 mM L-glutamine. After cells were allowed to attach for 4 hours, purified antibodies listed in FIG. 13 were added to final concentrations of 1.0, 3 and 10 μg/mL. 4D5 included as a positive control. The cells were allowed to grow for 3 days before cell growth was assessed by the XTT assay (Sigma) per the manufacturer's instructions. The difference in absorbance at 492 nm and 690 nm was taken as proportional to the number of cells in each well. These results (FIG. 13) suggest that D3.4 and to some degree D4.1 inhibit the growth of SKBR3 cells but not BT474. This difference in reactivity towards two cells lines with near equally high levels of HER2 expression may be explained by the fact that SKBR3 is know to shed greater levels of HER2 extracellular domain into the media and therefore may be more dependent on p95 retained in the cell.

US10273308B2. Methods of producing antibodies specific for p95 (2019-04-30). Laboratory Corporation of America Holdings [US]. Inventors: Jeff Sperinde [US], John William Winslow [US], Xueguang Jin [US], Gerald J. Wallweber [US].

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Cytotoxicity and Senescence Assay of DS-3032b

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Cells were seeded in triplicate onto 96-well plates, incubated for 24 h to permit adherence, then treated with 0 - 2000 nM DS-3032b for 24, 48 or 72 h. Cell viability was determined using the 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay (Sigma-Aldrich) according to the manufacturer's protocol. The IC50 was calculated using GraphPad Prism 6.0 (GraphPad Software). Cell proliferation was determined using the BrdU ELISA assay (Roche) according to the manufacturer's protocol. Senescence was measured using the fluorometric SA-β-gal activity assay (Cell Biolabs Inc.) according to the manufacturer's protocol and corrected for cell viability (XTT assay).

Arnhold V., Schmelz K., Proba J., Winkler A., Wünschel J., Toedling J., Deubzer H.E., Künkele A., Eggert A., Schulte J.H, & Hundsdoerfer P. (2017). Reactivating TP53 signaling by the novel MDM2 inhibitor DS-3032b as a therapeutic option for high-risk neuroblastoma. Oncotarget, 9(2), 2304-2319.

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Synthesis and Characterization of Chitosan-based Biomaterials

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Glucose, acetic acid, sodium hydroxide (NaOH), citric acid, ammonia, hydrochloric acid (HCl), potassium dichromate and ascorbic acid were delivered by POCH, Gliwice, Poland. Glucosamine, chitosan, cellulose, XTT assay and PBS (phosphate buffer solution) was delivered by Sigma-Aldrich, Poznań, Poland. Dulbecco’s modified Eagle medium (DMEM), streptomycin/penicillin and fetal bovine serum was delivered by Polgen, Łódź, Poland. Dialysis tubing (molecular weight cut off (MWCO) 500–1000 Da) was purchased from VWR, Gdańsk, Poland.

Janus Ł., Radwan-Pragłowska J., Piątkowski M, & Bogdał D. (2020). Facile Synthesis of Surface-Modified Carbon Quantum Dots (CQDs) for Biosensing and Bioimaging. Materials, 13(15), 3313.

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Evaluating Anti-Biofilm Peptides on Candida

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Example 5

The viability of various C. albicans biofilms (CA494, MYA 2876, CA 2519, CA 526 and ATCC 64124) in the presence of BmKn2, dBmKn2, Kn2-7 or dKn2-7 peptides was measured using the XTT assay (Sigma-Aldrich, St. Louis, Mo., USA) 24 hours after treatment. Results are depicted in FIGS. 3-7, and indicate that dBmKn2 and dKn2-7 caused 100% reduction in biofilm viability in all 5 fungal strains, and dKn2-7 showed up to a 4-fold higher efficacy against the fungal biofilms compared to the other three peptides. This indicates the D-amino acid forms of the peptides may be more stable against biofilm proteases. We note herein that the ATCC 64124 strain is ketoconazole resistant, and thus the data in FIG. 7 suggest that these peptides have activity against this drug resistant strain of C. albicans.

US10946065B2. Methods of treating fungal infections (2021-03-16). United States of America as represented by the Secretary of the Navy [US]. Inventors: Nancy Millenbaugh [US], Jeremy Wesley Gleaton [US], Dickson Kiprono Kirui [US].

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Radiation-Induced Cell Death Assay

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BMDMos were synchronized at G2/M by incubation with 100 nM nocodazole for 12 h. Thereafter, they were γ‐irradiated, then released and evaluated for cell death at indicated time points. Irradiation‐induced cell death was determined by XTT assay (Sigma‐Aldrich) according to the manufacturer's instructions. Absorbency was measured with a spectrophotometer (Tecan Infinite M200 Microplate Reader) at 450 nm with a reference wavelength at 650 nm. Relative number of dead cells as compared to the number of cells without treatment was expressed as percent cell death using the following formula: cell death (%) = 100% – 100% X(A450 of treated cells/A450 of untreated cells).

Jiang H., Xue X., Panda S., Kawale A., Hooy R.M., Liang F., Sohn J., Sung P, & Gekara N.O. (2019). Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death. The EMBO Journal, 38(21), e102718.

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Cell Viability and Colony Formation

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Cell viability assay was studied through XTT assay (Sigma-Aldrich) and trypan blue dye (Sigma-Aldrich) exclusion assay, where the cells that took up trypan blue were counted as dead. Cells stained with trypan blue and evaluated under a microscope using a hemocytometer. Soft agar colony formation was performed according to the previously described with minor modifications [15 ]. NucleoCounter^® NC-3000 (Chemometec, Denmark) was utilized for cell cycle analysis following the manufacturer’s instructions.

Wu Y.Y., Lai H.F., Huang T.C., Chen Y.G., Ye R.H., Chang P.Y., Lai S.W., Chen Y.C., Lee C.H., Liu W.N., Dai M.S., Chen J.H., Ho C.L, & Chiu Y.L. (2021). Aberrantly reduced expression of miR-342-5p contributes to CCND1-associated chronic myeloid leukemia progression and imatinib resistance. Cell Death & Disease, 12(10), 908.

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Starvation Effects on Cell Viability

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To evaluate whether repeated starvation steps and treatments would affect cell viability and consequently influence the measurement of glucose uptake, an XTT assay (Sigma–Aldrich) was initially performed. A total of 3x10³ cells/well belonging to Exp 1, 2, 4 and 6 derived from the 7 patients were plated in a 96-well plate and treated as previously described. Another experimental group was included as a control, consisting of cells continuously cultured in starvation medium (STARVED CTRL). The XTT assay was performed at the end of each day (T3, T6 and T7 sampling times) following the manufacturer’s instructions. The experiment was repeated thrice, and data are reported as mean ± SD over the three independent experiments.

Di Vincenzo M., Martino M., Lariccia V., Giancola G., Licini C., Di Benedetto G., Arnaldi G, & Orciani M. (2022). Mesenchymal Stem Cells Exposed to Persistently High Glucocorticoid Levels Develop Insulin-Resistance and Altered Lipolysis: A Promising In Vitro Model to Study Cushing’s Syndrome. Frontiers in Endocrinology, 13, 816229.

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