TNF-α and MCP-1 levels were measured using ELISA kits (R&D Systems, USA). The blood serum and cell supernatant were diluted and studied using a standard curve. All of the samples were measured in triplicate. The procedure was performed according to the manufacturer's instructions.
Anti-dsDNA antibody levels in the serum were determined by ELISA, as described previously (32 (link)). Briefly, 96-well microtiter plates (Costar) were pretreated with calf thymus dsDNA (Sigma-Aldrich) for 2 h at 37°C and then placed overnight at 4°C. After being washed with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h. After the plates were incubated with a 1:100 dilution of mouse serum, the levels of anti-dsDNA antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG2c, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, and anti-mouse IgE (all from Southern Biotech). Tetramethylbenzidine (TMB) substrate was used for the development, and absorbance at 450 nm was measured on a Thermo Multiskan Spectrum 1500.
Anti-dsDNA antibody levels in the serum were determined by ELISA, as described previously (32 (link)). Briefly, 96-well microtiter plates (Costar) were pretreated with calf thymus dsDNA (Sigma-Aldrich) for 2 h at 37°C and then placed overnight at 4°C. After being washed with PBS containing 0.05% Tween-20 (PBST), the plates were blocked with 1% BSA for 1 h. After the plates were incubated with a 1:100 dilution of mouse serum, the levels of anti-dsDNA antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG2c, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, and anti-mouse IgE (all from Southern Biotech). Tetramethylbenzidine (TMB) substrate was used for the development, and absorbance at 450 nm was measured on a Thermo Multiskan Spectrum 1500.
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