Enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was used to detect secreted growth factor, including NGF and VEGF, in the co-culture system. After 48 h of co-culture, 50 μl of supernatant of conditioned medium was harvested for the enzyme-linked immunosorbent assay, 40 μl of sample diluent was added to the sample well of the enzyme-labeled plate and 10 μl of the sample was used for testing. Then 50 μl of enzyme-labeled reagent was added to each well. After sealing the plate with a sealing film it was further incubated at 37°C for 30 min and washed five times with washing solution. Then, 50 μL of chromogenic fluid A was added to each well followed by 50 μL of chromogenic fluid B, shaken gently to mix and then developed in the dark at 37°C for 15 min. Finally, 50 μl of stop solution was added to each well to stop the reaction. The Benchmark-Plus Reader (Bio-Rad, Hercules, California, USA) was used to measure the absorbance of each well at 450 nm. Five parallel samples were set up for each group.