ALP activity in the seeded hBMSCs was measured after 7 and 21 days. Cell/scaffold constructs were sonicated with Triton-X 100 0.1% on ice followed by incubation at −80°C. Following two freeze-thawing cycles at −80°C, samples were incubated with working solution containing Sigma 104® phosphatase substrate (Sigma-Aldrich) and alkaline buffer solution (Sigma-Aldrich). Stop solution (1 M NaOH) was added, and the absorbance measured at 405 nm.
Sigma 104 phosphatase substrate
Manufactured by Merck Group
Sigma 104® is a phosphatase substrate used in various analytical and research applications. It serves as a colorimetric indicator for the detection and quantification of phosphatase enzyme activity. The substrate is hydrolyzed by phosphatases, resulting in the release of a colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline buffer solution, by Merck Group (3 mentions)
Triton x 100 buffer, by Merck Group (1 mentions)
Liquicolor kit, by EKF Diagnostics (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Alp staining kit, by Merck Group (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
5 protocols using sigma 104 phosphatase substrate
1
Quantifying ALP Activity in hBMSCs
2
Osteogenic Differentiation Analysis of ASCs and BMSCs
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ASCs and BMSCs in osteogenic and control media were fixed with paraformaldehyde 4% at day 3, 7 and 14 for ALP staining, using SIGMAFASTTM BCIP/NBT tablets (Sigma-Aldrich). Images of the staining were taken using the inverted microscope. For the ALP assay at day 14, ASCs and BMSCs were lysed in 0.1% Triton-X100 buffer (Sigma-Aldrich), followed by two freezing-thawing cycles at − 80 °C, after which 20 μl of cell lysate was added in 96-well plate and mixed with 90 μl of working solution containing Sigma 104® phosphatase substrate (Sigma-Aldrich) and alkaline buffer solution (Sigma-Aldrich). After incubation at 37 °C for 15 min, 50 μl of NaOH (sodium hydroxide) was added to stop the reaction. Absorbance was measured at 405 nm using the microplate reader. ALP activity assay was presented relative to BMSCs cultured in control medium as control samples.
3
Quantifying Osteogenic Differentiation via Mineralization and ALP
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Cells induced for osteogenesis were fixed in 60% isopropanol and stained with alizarin red (Rowley Biochemical, Danvers, MA) for evaluation of mineral deposition. The matrix calcium content was extracted with 0.5 M hydrochloric acid and quantified using the LiquiColor kit (Stanbio, Boerne, TX) following the manufacturer's instructions. Alkaline phosphatase (ALP) was also stained using an ALP staining kit (Sigma–Aldrich). The activity level of ALP in osteogenic BMSCs was determined by digesting cells with a buffer solution containing 2% Triton X‐100, 1.5 mM Tris base, 1 mM ZnCl2 and 1 mM MgCl2·6H2O at pH 9.0 for 30 minutes at 37°C and measuring the digestion solution at the adsorption wavelength of 405 nm after reacting with Sigma‐104 Phosphatase Substrate (Sigma–Aldrich). The results were subsequently normalized to the cell number determined by the total DNA content quantified by the Quant‐iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA), following the manufacturer's instructions.
4
IgG Antibody Binding to Tc24 by ELISA
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Binding of IgG antibody to immobilized Tc24 was measured in triplicate using an ELISA as previously descried.36 (link) In brief, bound mouse IgG was detected using a goat anti-mouse γ-chain specific AP-conjugated antibody. The reaction was developed by adding 100 μL of 1 mg/mL Sigma 104 Phosphatase substrate (Sigma-Aldrich Corporation) dissolved in 1 M diethanolamine, 0.5 mM MgCl2, pH 9.8 for 1 hour. The optical density was measured at 405 nm using a microplate reader (Molecular Devices, Menlo Park, CA).
5
Osteogenic Differentiation ALP Activity Analysis
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ALP activity after 3 and 10 days of culture in osteogenic medium was analyzed from the same Triton-X 100 lysates used in the proliferation test. Briefly, 90 µl of working solution containing 1:1 Sigma 104 ® phosphatase substrate and alkaline buffer solution (Sigma-Aldrich) was added to 20µl of each sample lysate in 96-well plates and incubated for 20 min at 37°C. After incubation, 50 µl of 1M NaOH was added to the wells to stop the reaction. Absorbance was measured at 405 nm. The results were normalized to the cell number determined by the proliferation test.
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