At D1 and D7, MSCs were lysed using 0.1% Triton x-100, 5 mm Tris-HCL pH8 solution. After three freeze/thaw cycles, the amount of double stranded DNA was measured in the supernatants, using a fluorescent Quant-iT Picogreen dsDNA Assay kit (Thermo Fisher). The amount of ALP was measured using SigmaFast™ p-Nitrophenyl phosphate (pNPP) tablets (Merck KGaA). A standard curve was established with serial dilutions of pNPP and a known quantity of ALP from bovine intestinal mucosa (Merck KGaA). A known amount of pNPP was added to each sample, prior to incubation, during 30 min at 37°C. The amount of product (p-nitrophenol) was determined by reading the absorbance at 405 nm, on a microplate reader, and the amount of ALP was quantified using the following equation: were A, pNPP in μmol; V, volume of sample in mL; T, time of incubation in minutes.
Sigmafast p nitrophenyl phosphate pnpp tablets
Manufactured by Merck Group
SigmaFast™ p-Nitrophenyl phosphate (pNPP) tablets are a substrate for the enzymatic detection and quantification of phosphatases. The tablets contain the chromogenic substrate p-nitrophenyl phosphate, which is hydrolyzed by phosphatases, resulting in the production of p-nitrophenol, a yellow-colored product that can be measured spectrophotometrically.
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Lab products found in correlation
2 protocols using sigmafast p nitrophenyl phosphate pnpp tablets
1
Quantifying MSC Alkaline Phosphatase
2
Quantifying Alkaline Phosphatase Activity
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At D1 and D7, MSCs were lysed using 0.1% Triton x-100, 5 mM Tris-HCL pH8 solution. After, three freeze/thaw cycles, the amount of double stranded DNA measured in the supernatants using a fluorescent Quant-iT Picogreen dsDNA Assay kit (Thermo Fisher), and the amount of ALP measured using SigmaFast p-Nitrophenyl phosphate (pNPP) tablets (Merck KGaA). A standard curve drawn with serial dilution of pNPP and a known quantity of ALP from bovine intestinal mucosa (Merck KGaA). Then, a known amount of pNPP added to each sample, prior to incubation during 30 min at 37°C. The amount of product (p-nitrophenol) determined by reading the absorbance at 405 nm on a microplate reader, and the amount of ALP quantified using the following equation:
ALP (U/mL) = A/V/T were A = pNPP in µmol, V = volume of sample in mL, T = time of incubation in minutes.
ALP (U/mL) = A/V/T were A = pNPP in µmol, V = volume of sample in mL, T = time of incubation in minutes.
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