Enzyme-linked immunosorbent assay (ELISA)
The contents of platelet-derived growth factor (PDGF)-BB, interferon (IFN)-γ and TGF-β1 (R&D Systems Inc., Minneapolis, USA) in the protein extracts were measured by ELISA according to the manufacturer’s instructions. Briefly, the protein extracts were lysed with 100 mM Tris (pH: 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 0.5% sodium deoxycholate. Then, 50 µL of standard, control, or sample was added to each well of the microplate, which was precoated with capture antibody; A second HRP-labeled antibody was added and bound to the antigen-antibody complex.Unbound detection antibody was washed away. Tetramethylbenzidine (TMB) substrate solution was added to the wells, and a blue color developed in proportion to the amount of analyte present in the sample. Color development was stopped, turning the color in the wells yellow. The absorbance of the color at 450 nm was measured. The contents of PDGF-BB, IFN-γ and TGFβ1 were calculated by means of standard curves.