E0227rb

Measurement of secretomes (TGF-β1, VEGF, FGF, and PDGF)/growth factor level in DD-CM were performed in each group using ELISA Assay (Bioassay Technology Laboratory, E0026Rb (VEGF); E0227Rb (FGF); E0133Rb (TGF-β1); E0052Rb (PDGF)). DD-CM was collected using sterile tubes. The cell culture supernatant was obtained from DD-CM, which had been centrifuged at 2000–3000 rpm for 20 minutes. Incubation was carried out for 60 minutes at 37°C using sample wells. The composition was a 40μL sample with 10μL of anti-secretome antibody, then 50μL of streptavidin-HRP. The next step was washing the palate using the 0.35 mL wash buffer for 30–60 seconds. This step was repeated 5 times. For automated washing, aspirate all wells and wash 5 times with a wash buffer. The wells were overfilled with a wash buffer. The plate was dried up with absorbent material like paper towels. Each well was given 50μL of the substrate solution A and then combined with 50μL substrate solution B. The plates were incubated for 10 minutes at 37°C in the dark. After incubation, 50μL Stop Solution was added to each well. Then set up Optical Density (OD value) of every well directly with a microplate reader set to 450 nm within 10 minutes (Bioassay Technology Laboratory, China).

Measurements of growth factor levels in LD-CM (secretome) in each group were done using an ELISA assay according to the laboratory protocol (Bioassay Technology Laboratory, E0052Rb (PDGF); E0133Rb (TGF-β1); E0026Rb (VEGF); E0227Rb (FGF)). The collection of LD-CM followed the instructions provided by the manufacturer. Within 10 min after adding the stop solution to each well (Bioassay Technology Laboratory), the optical density value was immediately measured using a microplate reader set to 450 nm.