Thiazolyl blue tetrazolium blue mtt assay

Manufactured by Merck Group

Sourced in Italy

Thiazolyl Blue Tetrazolium Blue (MTT) assay is a colorimetric method used to measure cell metabolic activity. It is based on the reduction of the yellow tetrazolium dye MTT to purple formazan crystals by metabolically active cells.

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Lab products found in correlation

Sigmafast p nitrophenyl phosphate tablets, by Merck Group (2 mentions) Infinite f500 reader, by Tecan (1 mentions) Bcip nbt kit, by Merck Group (1 mentions) Sigmafast p nitrophenyl phosphate kit, by Merck Group (1 mentions)

4 protocols using thiazolyl blue tetrazolium blue mtt assay

Cell Metabolism Quantification via MTT Assay

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Cell metabolism was measured by Thiazolyl Blue Tetrazolium Blue (MTT) assay (Sigma-Aldrich, Milan, Italy). The reagent was administered 0.5 mg/mL in P35/P60 dishes. After 3 h 30 min, the medium was removed, and cells containing the insoluble formazan were solubilized with 100% DMSO. Absorbance at 550 nm was then measured using a Tecan Infinite F500 reader.

Maffey A., Storini C., Diceglie C., Martelli C., Sironi L., Calzarossa C., Tonna N., Lovchik R., Delamarche E., Ottobrini L, & Bianco F. (2017). Mesenchymal stem cells from tumor microenvironment favour breast cancer stem cell proliferation, cancerogenic and metastatic potential, via ionotropic purinergic signalling. Scientific Reports, 7, 13162.

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Quantifying Alkaline Phosphatase Activity

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For microscopic views, we used the BCIP/NBT kit (Sigma) to detect alkaline phosphatase activity. Cells were washed once with Ca⁺⁺ and Mg⁺⁺-free PBS containing 0.05% Tween 20, then fixed with 10% formalin for 1 minute, and stained with BCIP/NBT substrate in the dark for 5–10 minutes. Afterwards, cells were washed once with the wash buffer, and pictures were taken. Positive staining is a dark blue color. To quantify the relative alkaline phosphatase activity, we used the SIGMAFAST™ p-Nitrophenyl Phosphate kit (Sigma) for AP measurement and the Thiazolyl Blue Tetrazolium Blue (MTT) assay (Sigma) for cell number.

He Y., de Castro L.F., Shin M.H., Dubois W., Yang H.H., Jiang S., Mishra P.J., Ren L., Gou H., Lal A., Khanna C., Merlino G., Lee M., Robey P.G, & Huang J. (2015). p53 Loss Increases the Osteogenic Differentiation of BMSCs. Stem cells (Dayton, Ohio), 33(4), 1304-1319.

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Osteogenic Cell Viability and Mineralization Assays

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Viability of osteogenic cells was measured using Thiazolyl Blue Tetrazolium Blue (MTT) assay (Sigma). Alkaline phosphatase (ALP) activity was quantified using SIGMAFAST™ p-Nitrophenyl phosphate Tablets (Sigma). For microscopic observation of ALP activity, SIGMA FAST™ BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) tablets were used according to manufacturer’s instructions. Mineralization was detected using 1% alizarin staining (pH 4.6). All reagents for measuring ALP activity, ALP visualization and mineralization were purchased from Sigma, MO, USA.

Suresh S., de Castro L.F., Dey S., Robey P.G, & Noguchi C.T. (2019). Erythropoietin modulates bone marrow stromal cell differentiation. Bone Research, 7, 21.

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Isolation and Characterization of Calvarial Osteogenic Cells

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Calvarial osteogenic cells were isolated from 6–8-week-old mice using previously described protocol. Proliferation of osteogenic cells was measured using Thiazolyl Blue Tetrazolium Blue (MTT) assay (Sigma). Alkaline phosphatase (ALP) activity was quantified using SIGMAFAST^™ p-Nitrophenyl phosphate Tablets (Sigma). For microscopic observation of ALP activity, SIGMA FAST^™ BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) tablets were used according to manufacturer’s instructions. Mineralization was detected using 1% alizarin staining (pH 4.6). All reagents for measuring ALP activity, ALP visualization and mineralization were purchased from Sigma, MO, USA.

Suresh S., Lee J, & Noguchi C.T. (2020). Erythropoietin signaling in osteoblasts is required for normal bone formation and for bone loss during erythropoietin stimulated erythropoiesis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(9), 11685-11697.

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