Tgf β1

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.

The corresponding ELISA kits were used to assess MDA (LifeSpan Biosciences; WA; USA; Cat.# LS-F28018), SOD (MyBioSource; CA, USA; Cat.# MBS036924), PAF (MyBioSource; CA, USA; Cat.# MBS2024376), TGF-β1 (MyBioSource; CA, USA; Cat.# MBS011634), HIF-1α (Kamia Biomedical; Seattle, USA; Cat.# KT-17920) and VEGF (MyBioSource; CA, USA; Cat.# MBS8506132). All steps were carried out in accordance with the manufacturers’ instructions. For the determination of protein content, the method of Lowry et al. (1951 (link)) was performed.

Monolayer of A549 cells were treated with 8 ng/ml of recombinant TGF-β1 (MyBioSource Inc, San Diego USA), and lysed at 6 hrs, 24 hrs and 48 hrs for RNA extraction. Nucleospin RNA extraction kit (Macherey-Nagel, Düren, Germany) was used to lyse for RNA extraction. Random hexamers primers were used for the reverse transcriptase polymerase chain reaction (RT-PCR) of 1 μg total RNA in a reaction volume of 20 μl; using Omniscript RT kit (Qiagen, Maryland USA). Real-time qRT-PCR was performed using the Quantitect Probe PCR kit with SYBER green reagent (Applied Bioscience, Foster City, CA USA).