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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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4 protocols using thiazolyl blue tetrazolium blue mtt assay

1

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.
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2

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.
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3

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.
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4

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.
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