Osteoblasts originating from the bone marrow (10,000 cells/well, Cosmo Bio) forming 80% confluent monolayers in the 6-well plates were cultured with stretching as described above and harvested by a cell scraper and solubilized in 100 μl of cell lysis buffer (Cell Signaling Technology, Danvers , MA). The lysate of the whole cell protein (1 mg/ml) was diluted 5-fold with 0.1 M carbonate buffer (pH 9.6) and placed on 96-well microtitration plates for 12 hr at 4°C. After blocking with 5% skimmed milk for 3 hr at room temperature, the plate was treated with 0.1 μg/ml hamster anti-podoplanin (AngioBio) for 3 hr at 20°C and with peroxidase-conjugated goat anti-hamster IgG (0.1 μg/ml) for 1 hr at 20°C, and then visualized by an ABTS peroxidase substrate system (SeraCare Life Sciences, Inc. [KPL]) in the plates at 37°C and absorbance changes at 405 nm were measured by a microplate reader. Cells treated with only a second antibody served as blanks. The amounts of podoplanin production in the cells were expressed as the mean absorbance of peroxidase metabolizing substrate of six wells.
Abts peroxidase substrate system
Manufactured by LGC
The ABTS peroxidase substrate system is a colorimetric solution used in horseradish peroxidase (HRP) enzyme-linked immunosorbent assay (ELISA) and other HRP-based applications. It consists of a ready-to-use substrate solution containing 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide. Upon reaction with HRP, the ABTS substrate is oxidized, producing a green-colored product that can be detected spectrophotometrically.
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2 protocols using abts peroxidase substrate system
1
Quantification of Osteoblast Podoplanin
2
Recombinant PEDV S Protein ELISA
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The recombinant S proteins were diluted to 2 μg/mL with coating buffer (KPL, SeraCare, MA, USA) and were coated on a Nunc maxi-soap plate (Thermo Fisher Scientific) at 4 °C for 16 h. After washing six times with washing buffer (KPL, SeraCare), each well was blocked with 300 μL blocking buffer (KPL, SeraCare) at room temperature for 1 h. The serum samples were diluted 40-fold in blocking buffer and then 100 μL was added per well to washed plates and incubated for 1 h at room temperature. After washing six times, as previously described, HRP-conjugated goat-anti-swine IgG antibody (KPL, SeraCare) was added to the wells at 1:1000 dilution and the plates were then incubated for 1 h at room temperature. The coloration procedure was initiated by applying 50 μL/well of ABTS® Peroxidase Substrate System (KPL, SeraCare) onto the washed plates, and the coloration step was stopped by adding 50 μL/well stopping solution (KPL, SeraCare). The signals were read at 405 nm using the EMax Plus Microplate Reader (Molecular Devices). In the ELISA assays, a double-positive sample for both immunostaining against PEDV-infected Vero cells and commercial PEDV ELISA was chosen as the inter-experimental positive control; similarly, a double-negative serum was chosen as the negative control in this study.
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