Xtt staining solution

Manufactured by Merck Group

Sourced in United States

The XTT staining solution is a laboratory reagent used for colorimetric cell viability assays. It is a tetrazolium-based compound that is reduced by metabolically active cells, resulting in the formation of a colored formazan product that can be measured spectrophotometrically.

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XTT solution (15 citations)

4 protocols using xtt staining solution

Cytotoxicity Assay of VX Exposure in NPC and MN

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NPC and MN were plated at 140,000 cells per well onto pre-coated poly-l-ornithine/laminin 24-well plates and exposed to 400 µM VX, 600 µM VX, 800 µM VX, 1000 µM VX or ACN alone (solvent control) in NMM (3 wells each) as described above. After incubation for 5 days, cells were washed once with PBS. XTT staining solution (Sigma-Aldrich, St. Louis, USA) was prepared freshly by mixing 100 µL coupling reagent and 5 mL XTT labelling reagent. After incubating the cells for 3 h with 400 µL NMM and 200 µL XTT staining solution in a humidified atmosphere at 37 °C containing 5% (v/v) CO₂, the absorbance was quantified at 450 nm and a reference wavelength at 630 nm. After elimination of background absorbance determined in the wells containing only NMM, the viability was normalized to solvent controls. The experiment was carried out three times independently (biological replicates) in three technical replicates per concentration.

Schaefers C., Schmeißer W., John H., Worek F., Rein T., Rothmiller S, & Schmidt A. (2024). Effects of the nerve agent VX on hiPSC-derived motor neurons. Archives of Toxicology, 98(6), 1859-1875.

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Evaluating Drug Resistance in Cell Cultures

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For resistance determination, 40,000 cells were seeded per well in a clear bottom-flat 24-well plate (Greiner) including medium control as blank and were grown overnight. The cells were exposed to increasing concentrations from 0.2 to 500 μM SM including vehicle control for 5 days. Glutathione S-transferase (GST) inhibitors ezatiostat (TLK199, Cayman Chemical) and ethacrynic acid (Sigma) were added to medium in final concentrations of 400 nM and 600 nM, respectively. Stock solutions of both inhibitors were prepared in DMSO with 10 mg/mL. After pre-dilution in 100 % EtOH, GST activator ethoxyquin (Sigma) was used with 24 μM final concentration as a pre-treatment for 24 hours. For testing resistance against H₂O₂, concentrations between 0.24 and 80,000 μ M were used including medium control for 5 days. Therefore, 30 % H₂O₂ (Sigma) was pre-diluted in sterile ddH₂O. After exposition over 5 days, cells were washed with medium and XTT staining solution (Sigma) was prepared by mixing 5 mL XTT labeling reagent with 0.1 mL electron-coupling reagent per plate. 400 μ L medium with 200 μ L of XTT staining solution were added per well and incubated for 6 h. Extinction was measured at 450 nm with reference at 630 nm. All conditions were tested in biological triplicates.

Rothmiller S., Schröder S., Strobelt R., Wolf M., Wang J., Jiang X., Worek F., Steinritz D., Thiermann H, & Schmidt A. (2017). Sulfur mustard resistant keratinocytes obtained elevated glutathione levels and other changes in the antioxidative defense mechanism. Toxicology letters, 293, 51-61.

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Senolytic Drug Screening using XTT

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Non-senescent and senescent cells were seeded at 6,735 cells per well in a 96-well plate (Greiner AG, Kremsmünster, Austria) including medium controls each for background determination. The following day, the cells were exposed to increasing concentrations of senolytic drugs shown in Table S2 as well as solvent controls for 5 d or 24 h. Then cells were washed once with PBS and XTT staining solution (Sigma-Aldrich, St. Louis, USA) was prepared by mixing 5 mL XTT labeling reagent with 100 µL electron-coupling reagent per plate. Cells were incubated with 100 µL medium and 50 µL XTT staining solution and absorbance was determined at 450 nm with the reference set to 630 nm. Background absorbance was removed using wells only containing medium and viability was normalized to solvent controls. All experiments were performed with 4 biological replicates per group and three times (except 17-DMAG and dasatinib 24 h each two times) independently.

Rothmiller S., Jäger N., Meier N., Meyer T., Neu A., Steinritz D., Thiermann H., Scherer M., Rummel C., Mangerich A., Bürkle A, & Schmidt A. (2021). Chronic senescent human mesenchymal stem cells as possible contributor to the wound healing disorder after exposure to the alkylating agent sulfur mustard. Archives of Toxicology, 95(2), 727-747.

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Hydrogen Peroxide Cytotoxicity Assay

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MSCs were plated at 40,000 cells per well in two 24-well plates (Greiner AG, Kremsmünster, Austria) including medium control and grown overnight. The next day, cells were exposed to increasing concentrations of H₂O₂ (0.2–80,000 µM) as well as solvent control for 5 days. The 30% (w/w) H₂O₂ solution in H₂O (Sigma-Aldrich, St. Louis, USA) was pre-diluted in ultra-pure water and finally diluted in culture medium. Afterwards cells were washed once with PBS and XTT staining solution (Sigma-Aldrich, St. Louis, USA) was prepared by mixing 5 mL XTT labeling reagent with 100 µL electron-coupling reagent per plate. Cells were incubated with 400 µL medium and 200 µL XTT staining solution, and absorbance was determined at 450 nm with a reference set at 630 nm. Background absorbance was removed using wells only containing medium and viability was normalized to solvent controls. This experiment was performed six times independently (i.e., with cells obtained from six individual donor materials).

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