Pdgf bb

The PRF membranes were cut into 6 × 6 mm pieces with a 6-mm biopsy punch and homogenized using homogenizing pestles. After centrifuging at 3000 rpm for 10 min at 4 °C, the supernatant was collected and the concentration of PDGF-BB was measured using ELISA kits according to the manufacturer’s protocol (Mybiosource, San Diego, CA, USA): PDGF-BB (Cat. No. MBS0033120).

To measure growth factor release, both A-PRF preparations were transferred into a 6-well plate. One milliliter of DMEM (Gibco, Life Technologies Corporation, New York, NY, USA) was added to each sample. The plate was then placed in a 5% CO₂ incubator at 37 °C to allow for growth factors to be released into the culture media during the three-day period of the study. At 1 h, 24 h, and 72 h, 1 mL of the culture media was collected, kept at −20 °C, and replaced with 1 mL of additional fresh culture media. The collected samples at each time point were analyzed for canine TGF-β1, VEGFA, and PDGF-BB using commercial ELISA kits, including TGF-β1 (MyBioSource, Inc., San Diego, CA, USA), VEGFA (Abcam, Cambridge, UK), and PDGF-BB (Abcam, Cambridge, UK), respectively, according to the manufacturers’ instructions. Optical density was assessed using a microplate reader at 450 nm. The absorbance measured at 630 nm was subtracted from that measured at 450 nm for an optical density correction. The measurement was performed in duplicate.