C3H10T1/2 embryonic murine fibroblasts (ATCC) were maintained in growth medium (GM) consisting of Eagle’s minimum essential medium (ATCC) supplemented with 10% fetal bovine serum (FBS, Hyclone) in a 37°C humidified incubator with 5% CO2. Between passages 5 and 15, cells were plated in 96-well plates at an initial seeding density of 3,200 cells/well in GM supplemented with 20 mM HEPES (Lonza). The cells were allowed to proliferate for three days after seeding, and then the media was replaced with fresh GM supplemented with 20 mM HEPES, 2% FBS, and various treatments containing Shh or mvShh. After an additional four days, the cells were rinsed once with TBS (50 mM Tris-Cl, pH 7.6; 150 mM NaCl), and after aspirating the TBS, the cells were flash frozen at −80°C. 0.1% Triton-X 100 in TBS was then added to each well and then the plate was agitated for 30 minutes to thaw and lyse the cells. ALP enzyme activity in each well was detected using the p-nitrophenyl phosphate (pNPP) liquid substrate system (Sigma) and normalized to the total protein concentration measured by the BCA assay. We fit the resulting dose-response curves using four-parameter logistic non-linear curves to determine the EC50 for each treatment.
P nitrophenyl phosphate pnpp liquid substrate system
Manufactured by Merck Group
Sourced in United Kingdom
P-Nitrophenyl Phosphate (pNPP) Liquid Substrate System is a colorimetric solution used for the detection and quantification of various enzymatic activities. It contains the substrate p-nitrophenyl phosphate, which undergoes a color change upon enzymatic hydrolysis, allowing for the measurement of enzyme concentration or activity.
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5 protocols using p nitrophenyl phosphate pnpp liquid substrate system
1
Alkaline Phosphatase Activity in Murine Fibroblasts
2
Alkaline Phosphatase Activity Assay in MC-3T3-E1 Cells
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MC-3T3-E1 cells were cultured in DMEM medium at an initial density of 2×104 cells/well in 24-well culture plates and collected from each well after 3 days ES stimulation as described above. After the different frequency ES stimulation, the cells were assayed for ALP activity with a p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma) following the manufacturer’s instructions. First, cells were lysed in a lysis buffer (Sangon) and incubated for 30min at 37°C and centrifuged at 12,000g force for 10min at 4°C. The clear cell lysis was transferred into a new 1.5ml centrifuge tube for the following ALP assay. Add 10μl clear cell lysis into a 96-well transparent plate. Add 200 μl of pNPP solution to each well. Incubate the plate in the dark for approximately 30 minutes at room temperature. After the incubation period, read the plate at 405 nm on a multiwell plate reader (Tecan M200). The enzymatic activity was expressed as mmoles of p-NP/min/mg Protein.
3
Osteogenic Differentiation of hMSCs
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hMSCs were seeded onto a 24 well plate at a density of 10 000 cells/well in triplicate. Cells were incubated in MSC growth medium until 90% confluent. The MSC growth medium was then replaced with Mg filtered medium or non-filtered medium (DMEM) supplemented with 10% (v/v) FBS, L-glutamine final media concentration 2 mM, and 100 units/ml penicillin-streptomycin and cultured for a period of 12 days. Cells cultured in MSC growth medium were used as the standard control and cells cultured in osteogenic medium were used as the positive control. ALP assay was performed at day 2, 7 and 12 using p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System, (Sigma Aldrich, UK). Briefly, cells were lysed in 0.1% Triton X-100; pNPP solution was added to each well and the plate was incubated in the dark for approximately 30 minutes at room temperature. The production of p-nitrophenol (PNP) was quantified at an absorbance of 405 nm using a plate reader and normalised to DNA concentration.
4
Allergen-specific IgE Detection Assay
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MicroWell 96-Well Microplates (Nunc, Thermo Fisher Scientific) were coated with either nHel a 6 (5 ng/μl) or sunflower pollen extract in a bicarbonate buffer (pH 9.2) followed by blocking with 3% BSA (Sigma). Wells were then exposed to patient sera in 1:5 v/v at 4 °C for 16 h. Bound IgEs were detected by Monoclonal Anti-Human IgE−Alkaline Phosphatase antibody produced in mouse (Sigma-Aldrich, Catalogue no. A3076) at 1:1000 v/v and p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma-Aldrich). The reaction was stopped with 3 N NaOH, and absorbance was measured at 405 nm. An individual patient serum having P/N value [ratio between A405 of a patient serum (P) and the mean of healthy controls (N)] more than 2.0 was considered as positive for that allergen.
5
ALP Activity Quantification in MC-3T3-E1 Cells
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MC-3T3-E1 cells (5 × 104/well) were seeded onto scaffolds and cultured in high-glucose DMEM. After 7 days, the ALP activity of one group was assayed using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium ALP colour development kit (Beyotime, China). ALP-positive cells were captured under a light microscope (Olympus IX71) in five randomly selected fields. Data are presented as mean ± standard deviation (SD), with n = 3. A cell lysate kit (Beyotime, China) was used in another group of cells to extract the total proteins in accordance with the specifications. ALP activity was assayed with a p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma) following the manufacturer’s instructions. A total of 200 µl pNPP solution was added to each well containing 10 µl cell lysate. The plates were incubated at room temperature for 30 min and activities were detected and read at a wavelength of 405 nm (Thermo MK3). The protein concentration of cell lysates was quantified using the BCA Kit (Beyotime, China). ALP enzyme activity was calibrated using protein concentration.
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