Vegf elisa kit

For quantification of VEGF release in cell lysates, the cells (5x10⁴/well) were seeded in 96-well plates with DMEM. After discarding the supernatant, the cell lysate was prepared as described for the HIF-1α ELISA. ELISA (kit sensitivity, 20 pg/ml) was performed using a VEGF ELISA kit (Boster Biological Technology; cat. no. EK0540) according to the manufacturer's protocol. Briefly, 100 µl of the samples and standards were added to 96-well plates pre-coated with the capture antibody. After incubation for 90 min at 37˚C, the supernatant from each well was decanted and 100 µl of the prepared biotinylated primary antibody mixture was added. After 1 h of incubation at 37˚C, all wells were washed 3 times with 300 µl 0.01 M PBS. The wash buffer was discarded and 100 µl of the prepared secondary antibody, Avidin-Biotin- Peroxidase Complex, was added to each well and incubated for 30 min at 37˚C. Subsequently, all wells were washed 5 times with 300 µl 0.01 M PBS. After removal of the washing buffer, 90 µl color developing agent was added to each well and incubated at 37˚C for 25 min. Finally, 100 µl stop solution was added to each well and the plate was read at 450 nm using an UV microplate reader.

Second passage hUCMSCs were seeded in DMEM/F12 containing 10% fetal bovine serum. After the cells reached 90% confluence, the medium was replaced with DMEM/F12 medium containing 1% fetal bovine serum, followed by culture for an additional 48 hours at 37°C in a humidified 5% CO₂ incubator. The hUCMSC-conditioned medium was collected, filtered through a 0.22-μm filter, and immediately frozen at −80°C until use. DMEM/F12 medium containing 1% fetal bovine serum was used as a control. The conditioned media were analyzed using human brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), neurotrophin-3 (NT-3), basic fibroblast growth factor (bFGF), nerve growth factor (NGF)-β and vascular endothelial growth factor (VEGF) ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocols. All samples were analyzed in triplicate, and the optical density was measured at 450 nm (Wellscan MK3, Labsystems Dragon, Helsinki, Finland).