P nitrophenol phosphate

The enzymatic activity of alkaline phosphatase (ALP) was determined to check osteogenic differentiation of PDL cells during the different culture conditions (DA, SA, DR, and SR). After culture, cell lysates from each sample were achieved by freeze-thaw cycles three times and followed by addition of cell lysis buffer. The samples lysates were moved to the tubes and then centrifuged, and the collected cell lysates were used for measurements. The samples (50 μl) were added to 50 μl of working reagent containing equal parts (1:1:1) of 1.5 M 2-amino-2 methyl-1-propanol (Sigma, A5888), 20 mM p-nitrophenol phosphate (Sigma, P4744), and 1 mM magnesium chloride (Sigma, 208337). The samples were then incubated, and the reaction was stopped with the addition of 100 μl of 1 N NaOH on ice. The p-nitrophenol produced by the reaction was then determined based on the absorbance at 405 nm using micoplate-reader (iMark, BioRad, CA, US). The reading for the sample was calculated from a standard ALP activity curve prepared using p-nitrophenol stock standard (Sigma, N7660). Cell proliferation was measured by qualify dsDNA via PicoGreen fluorescence assay to standardize ALP production measurements. The ALP specific activity was calculated by normalizing the ALP activity to dsDNA produced from each sample. Each test was performed on three replicate samples (n = 3).

Shh proteins were expressed in Bosc23 cells and secreted into the supernatants. Supernatants were added to C3H10T1/2 osteoblast precursor cells and their Hh-dependent differentiation was used as a read-out to determine Hh bioactivity. Prior to analysis, protein aliquots were immunoblotted to confirm comparable protein amounts and protein integrity before use in the activity assay. The remaining conditioned media were then sterile filtered and applied to C3H10T1/2^{85 (link)} cells in 15 mm plates. Cells were lysed 5 days after induction (20 mM HEPES, 150 mM NaCl, 0.5 % Triton X-100, pH 7.4) and the amount of alkaline phosphatase produced as a response of Shh-induced and Shh^K/A-induced osteoblast differentiation was measured at 405 nm with 120 mM p-nitrophenolphosphate (Sigma) in 0.1 M glycine buffer at pH 8.5. All assays were performed in triplicate.

C3H10T1/2 cells were grown in DMEM supplemented with 10% FCS and antibiotics. Twenty-four hours after seeding, Shh-conditioned media was mixed 1:1 with DMEM containing 10% FCS and antibiotics, and applied to C3H10T1/2 cells in 15 mm plates. In some cases, 0–6 ng of double-lipidated, HEK293-derived human Shh (R&D Systems, 8908-SH) served as positive activity controls during the assays. To some samples, 2.5 μM cyclopamine, a specific inhibitor of Shh signaling, and 1 µg/mL of Shh neutralizing antibody 5E1 [29 (link)] were added to confirm the specificity of the assay. In general, due to variable expression levels, mutant and wild-type proteins were adjusted to similar levels prior to the induction of C3H10T1/2 differentiation, as determined by Western blotting of an aliquot of the same stock. Cells were lysed 5–6 days after induction (20 mM Hepes, 150 mM NaCl, 0.5% TritonX-100, pH 7.4) and osteoblast-specific alkaline phosphatase (Alp1) activity was measured at 405 nm after the addition of 120 mM p-nitrophenol phosphate (Sigma) in 0.1 M glycine buffer, pH 9.5. Assays were always performed in triplicate. Cells were authenticated and tested negative for mycoplasma.

2 µg of protein were used to colorimetrically (OD 405nm) measure alkaline phosphatase specific activity with 5 mM p-nitrophenolphosphate (Sigma-Aldrich) used as a substrate. Cell lysates were incubated in 2 mM p-nitrophenol phosphate for 30 min at 37°C. The reaction was stopped by adding 1 M NaOH, and the product was quantified at 405 nm. One unit of alkaline phosphatase was defined as 1 µmol substrate hydrolyzed per hour (perµg protein/sample).

Osteoclasts on glass coverslips were fixed with 4% paraformaldehyde, and stained for TRAP activity (Sigma) according to the manufacturer's manual. Images were captured using a Nikon Diaphot 300 inverted microscope. TRAP activity was quantified by a colorimetric method. Cells grown in 48-well plates were washed with Hanks Buffer and incubated at 37°C with a pre-warmed, 200 μL mixture containing 0.1% SDS, 2 mg p-nitrophenol phosphate, acetate and tartrate solution (Sigma) for 30 min. The reaction was stopped by adding 40 μL of 0.5 M NaOH and absorbance was read at 405 nm in a microplate reader (BMG Labtech, Cary, NC).

ctDNA was passed through an Millex-HA 0.45 mm filter (Millipore) to eliminate ssDNA. Indicated amounts of ctDNA or supercoiled plasmid DNA were incubated with recombinant human topo I in 40 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.5 mM EDTA, 30 µg/ml BSA for 30 min at 37 °C. Then, 30 µl of sample per well was used for coating a 384-well Nunc MaxiSorp plates overnight at 4 °C. Plates were blocked with PBS containing 1% casein (Pierce) for 1 h at 37 °C, and plates were incubated with monoclonal anti-dsDNA abs at a concentration of 1 µg/ml for 1 h at 37 °C. Subsequently, plates were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotech) for 1 h at 37 °C and developed with p-nitrophenol phosphate (Sigma-Aldrich). Optical density (OD) was measured at 405 nm with a reference filter at 490 nm. When used oligonucleotides for these experiments, forward and reverse strands were annealed for 3 min at 93 °C. After incubation with recombinant topo I as described above, modified oligonucleotides were attached to Nunc MaxiSorp plates overnight at 4 °C using DNA Coating Solution from Pierce. Detection of anti-dsDNA binding was performed as described above.