Mouse anti human tgf β3

The amount of TGF-β3 release from the growth factor loaded cartilage ECMderived scaffold was determined via ELISA, as previously described [7, 22] . 96 well plates were coated with capture antibody (360 µg ml -1 ) with mouse anti-human TGF-β3 (R&D Systems, UK). The samples (8 time points) and TGF-β3 standards (ProSpec-Tany TechnoGene Ltd, Israel) were incubated for 2 hours. After washing and drying, detection antibody (18 µg ml -1 of biotinylated goat anti-human TGF-β3) was added to the plate and incubated (2 hours). The next step was to wash, dry and incubate the plate in streptavidin-HRP (horseradish-peroxidase) (R&D Systems, UK) for 20 minutes in the dark. Substrate solution (1:1 mixture of H 2 O 2 and tetramethylbenzidine; R&D Systems, UK) was added to each well, followed by incubation (20 minutes) avoiding direct light. Stop solution (2 N H 2 SO 4 ; Sigma-Aldrich, Germany) was added and the optical density was determined immediately with a plate reader set to 450 nm.

The TGF-β3 present in the ECM-derived scaffolds before seeding with FPSC was measured using a previously reported protocol 19 . Briefly, the scaffolds were treated with a solution of 4 M guanidine hydrochloride (Pierce) at 4°C for 2 days to extract any growth factors. TGF-β3 content was determined by ELISA 6c, [19] [20] . By following manufacturer instructions, capture antibody (360 µg ml -1 ) was in 96 well plates (mouse anti-human TGF-β3; R&D Systems, UK). The samples and standards (ProSpec-Tany TechnoGene Ltd, Israel) were incubated (2h). Furthermore, detection antibody (18 µg ml -1 of biotinylated goat antihuman TGF-β3) was added and incubated (2h). Next, the plate was washed, dried and incubated in streptavidin-HRP (horseradish-peroxidase; R&D Systems, UK). Substrate solution (1:1 mixture of H 2 O 2 and tetramethylbenzidine; R&D Systems, UK) was added to each well, followed by incubation (no light). Stop solution (2N H 2 SO 4 ; Sigma-Aldrich) was added and the optical density was determined (450 nm).