Alkaline Phosphatase (ALP) is one of the most reliable markers for osteogenic differentiation, since it is produced by osteogenic cells such as osteoblasts [34 (link), 35 (link)]. The scaffolds were washed with 1x PBS and then kept at -80°C in dry conditions until further use. The frozen MSC-seeded scaffolds underwent homogenization using a Polytron PT 1200 E benchtop homogenizer (Kinematica, Lucerne, Switzerland) in 1% Triton buffer (Sigma Aldrich, Steinheim, Germany). The homogenate was centrifuged and the supernatant was added to a solution of para-Nitrophenylphosphate (p-NPP) (Sigma Aldrich, Steinheim, Germany) for 90 min. The ALP converts p-NPP to para-Nitrophenol (p-NP) causing a change of color to yellow. The extinction of p-NP which corresponds to the ALP activity was measured photometrically at 405 nm with a reference wavelength of 490 nm using a MRX Microplate Reader (Dynatech Laboratories, Stuttgart, Germany). The ALP activity was normalized to the dsDNA content of each sample, since the amount of dsDNA is directly correlated with the number of cells.
Para nitrophenylphosphate pnpp
Manufactured by Merck Group
Sourced in United States, Germany
Para-nitrophenylphosphate (pNPP) is a colorless, crystalline compound used as a substrate in various laboratory assays. It serves as a chromogenic substrate for the detection and quantification of enzymatic activity, particularly phosphatases. When hydrolyzed by the enzyme, pNPP produces a yellow-colored product, para-nitrophenol, which can be measured spectrophotometrically.
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Lab products found in correlation
Triton buffer, by Merck Group (1 mentions)
3 n naoh, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Wuhan Boster Biological Technology (1 mentions)
Laminin, by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Elisa reader, by Molecular Devices (1 mentions)
Tissue culture reagents, by Thermo Fisher Scientific (1 mentions)
Monoclonal anti v5, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Benzaldehyde, by Merck Group (1 mentions)
Nitroblue tetrazolium chloride, by Merck Group (1 mentions)
Potassium superoxide, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by PAN Biotech (1 mentions)
4 dimethylaminopyridine, by Merck Group (1 mentions)
18 crown 6 ether, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
N butyllithium, by Merck Group (1 mentions)
2 7 dichlorofluorescein, by Merck Group (1 mentions)
Diisopropylamine, by Merck Group (1 mentions)
25 protocols using para nitrophenylphosphate pnpp
1
Quantification of Osteoblast Differentiation
2
Osteogenic Differentiation of hMSCs
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The hMSCs were seeded onto the experimental specimen in a 24-well plate to a density of 20,000 cells/well. After 1 h of incubation, visible light irradiation (470 and 600 nm) was performed for 30 min. Additional visible light irradiation and fresh osteogenic media change was conducted every three days. After one and two weeks of cultivation, the cultured cells were collected and lysed in a lysis buffer solution (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40). After 30 min of lysis, 200 µL of para-nitrophenyl phosphate (pNPP; Sigma, MO, USA) was added to 50 µL cell lysate and reacted at 37 °C. After 30 min of p-NPP reaction, 50 µL of 3 N NaOH (Sigma, MO, USA) was added to the solution to stop the reaction. After measuring the absorbance at 405 nm using the microplate ELISA reader, the enzyme activity per unit protein content was calculated by dividing the absorbance value by the average value of the total protein amount. Four specimens were collected at a time to form one sample in order minimize the experimental errors. The number of samples used in the statistical analysis is three.
3
Quantifying Laminin Binding Capacity
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In order to measure laminin binding capacity, a modified enzyme-linked immunosorbent assay (ELISA) technique was employed in which laminin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was added to 96-well plates (Costar). The plates were then incubated at 4°C for 24 h, and ventilated to dry. After washing the plates with phosphate-buffered saline (PBS; pH 7.3) five times, the plates were blocked with 2.5% (wt/vol) bovine serum albumin (Wuhan Boster Biological Technology, Ltd.- Wuhan, China) containing 0.1% (vol/vol) Tween 20 at 37°C for 2 h. The four recombinant proteins with increasing concentrations (0–4 µM) were added to the plates (100 µl/well) and incubated at 37°C for 2 h. The plates were then washed five times with PBS to remove redundant proteins. Proteins bound to laminin were tested using mouse antibody against polyhistidine (cat. no. H1029; 1:1,000 dilution; Sigma-Aldrich) for 1 h at 37°C, followed by goat antibody to mouse IgG (alkaline phosphatase-conjugated; cat. no. P7998; 1:10,000; Sigma-Aldrich) for 1 h at 37°C. Bound proteins were detected using the alkaline phosphatase (AP) reaction with para-nitrophenylphosphate (pNPP; Sigma-Aldrich) in AP buffer (pH 9.6) for 15 min at 37°C; this reaction was stopped using 3 M NaOH. Optical density at 405 nm (OD405) was quantified using an ELISA reader (Molecular Devices, LLC, Sunnyvale, CA, USA).
4
Age-Dependent STEP Expression in Rats
5
PTP1B Inhibition Assay Protocol
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N-Methyl-4-piperidone, Benzaldehyde, Diisopropylamine, n-Butyllithium (2.5 M solution in hexanes), 4-(Dimethylamino)pyridine (DMAP), and Sodium bicarbonate were purchased from Merck. Acetic acid, Dichloromethane, Tetrahydrofuran, Ethanol, Ethyl acetate, Citric acid, Sodium Sulfate(VI), and Acetic anhydride were purchased from POCH, Gliwice, Poland. Vanilin was purchased from Acros, Waltham, Massachusetts, USA. Dimethyl-sulfoxide (DMSO), Potassium superoxide, 18-crown-6-ether, Nitro blue tetrazolium chloride (NBT), Curcumin, Fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), recombinant PTP1B phosphatase, para-nitrophenyl phosphate (pNPP), and 2′,7′-dichlorofluorescein were purchased from Sigma-Aldrich, Saint Louis, Missouri, USA. Dulbecco’s Modified Eagle’s Medium (DMEM) and Phosphate-buffered saline (PBS) were purchased from PAN-biotech, Aidenbach, Germany. Lastly, 4–20% MP TGX Tain-Free Gel 10W was purchased from Bio-Rad Laboratories, Hercules, CA, USA and PTP1B antibodies were purchased from Cell Signaling, Danvers, MA, USA.
6
Synthetic peptide ELISA protocol
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Synthetic citrulline-, arginine- and homo-citrulline-containing peptides were from Schafer-N (Lyngby, Denmark). Streptavidin, alkaline phosphatase (AP)-conjugated goat anti-human IgG and para-nitrophenylphosphate (pNPP) were from Sigma Aldrich (St. Louis, Mo, USA). Tris–Tween–NaCl (TTN) buffer (0.05 M Tris, 0.3 M NaCl, 1% Tween 20, pH 7.4) and AP-substrate buffer (1 M diethanolamine, 0.5 mM MgCl2, pH 9.8) were from SSI Diagnostica (Hillerød, Denmark).
7
Quantifying PAR2 Receptor Cleavage
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Secreted epithelial alkaline phosphatase (SEAP)–tagged versions of human PAR2 (UniProtKB accession P55085 ; fused at Q27) protein as well as the canonical cleavage site mutant R36G were generated to allow quantification of receptor cleavage (Ludeman et al., 2004 (link)). PAR1 (F2r) knockout mouse lung fibroblasts (KOLFs; Trejo et al., 1996 (link)) were grown in DMEM supplemented with antibiotics and 5% serum. Transfection was performed with TransitLT1 transfection (Euromedex) in complete medium as recommended. 48 h after transfection, cells were washed with Opti-MEM for 1 h and incubated for one additional hour in Opti-MEM with or without the respective MC-specific proteases (500 ng/ml), and supernatants were collected. A second 20-min incubation with 10 nM trypsin stripped all remaining SEAP moiety (as verified in separate experiments) from the cell surface. SEAP activity in the conditioned media was determined at OD405 after hydrolysis of para-nitrophenyl phosphate (pNPP; Sigma-Aldrich). This permitted calculation of percentage of surface receptors cleaved during the initial 60-min incubation.
8
Quantifying ALP Activity in hPDLSCs
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ALP activity of hPDLSCs of each specimen was assessed after differentiation for 14 days using a commercially available kit (LabAssayTM kit, Wako Pure Chemicals, Tokyo, Japan) according to the manufacturer’s procedures. Wells were washed to remove unattached cells, after which 0.1% Triton in 1 mL PBS was added. The cell layers were detached and then sonicated for 30 s. Para-Nitrophenylphosphate (p-NPP) (Sigma Aldrich, Steinheim, Germany) was added to the supernatant for 90 min after the removal of cell debris by centrifugation. ALP transforms p-NPP to yellow para-Nitrophenol (p-NP), which was measured photometrically at 405 nm [25 (link)].
9
Quantifying Secreted Alkaline Phosphatase
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The SEAP (human-secreted alkaline phosphatase) activity assay was performed in parallel for the supernatant and the lysed cell pellets of a harvested cell culture. A total of 100 μL of lysate and 100 μL of supernatant were pipetted into round-bottom plates for the heat inactivation of endogenous phosphatases at 65 °C for 1 h. Afterward, 80 μL of the lysate and supernatant samples was transferred into transparent 96-well flat-bottom plates and mixed with 100 μL of SEAP buffer (20 mM L-homoarginine, 1 mM MgCl2, and 21% (v/v) diethanolamine). Before the determination was started, 20 μL of 120 nM para-nitrophenylphosphate (pNPP, Sigma Aldrich, Darmstadt, Germany) was added. The absorbance was measured in a Berthold technologies Tristar2S LB942 Multimode plate reader for 2 h at 405 nm as previously described [19 (link)].
10
Development of Mycotoxin Detection Assay
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DELFIA series buffers, streptavidin and rabbit anti-mouse-coated microtiter plates were purchased from Kaivogen Diagnostics (Turku, Finland). All measurements were done with a Victor 1420–fluorometer from Perkin-Elmer (Turku, Finland). The magnetic nanoparticles and magnetic bead concentrator were purchased from Dynal (Norway). The mycotoxins nivalenol (NIV), Deoxynivalenol (DON), 3-Acetyldeoxynivalenol (3-AcDON), 15-Acetyldeoxynivalenol (15-AcDON), T-2 toxin and HT-2 toxin were purchased from Biopure Guntramsdorf, Austria). Hyperphages were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The E. coli cell lines used for the sorting and expression of the antibody libraries were purchased from Stratagene (La Jolla, CA, USA): BL21 (F-, dcm, ompT, hsdS[rB− mB−], gal [malB+], K-12[λS]) and XL1-Blue (recA1, endA1, gyrA96, thi-1, hsdR17, relA1, lac [F`, TetR]). All microbiological reagents were prepared as described in Sambrook et al. [16 ]. The single-chain alkaline phosphatase (scFv-ALP) fusion proteins were purified with HisPur Ni-NTA spin columns Thermo Fisher Scientific (Waltham, MA, USA). The ELISA substrate para-nitrophenylphosphate (pNPP) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
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