Human tenascin c

ECM-mimetic hydrogels (50 μl) were generated from a HEPES solution (20 mM, 150 nM NaCl, pH 7.4) containing 8 mg/ml human fibrinogen (Enzyme Research Laboratories), 1 mg/ml human plasma fibronectin (Sigma), 500 μg/ml human vitronectin (Peprotech), 50 ug/ml human tenascin C (R&D Systems), 50 μg/ml heparan sulfate (Sigma) and 500 ng/ml of PDGF-BB or IL-1Ra variants. Matrices were polymerised in Ultra-Low Cluster 96-well plate (Corning) at 37 °C for 2 h with 10 U/ml bovine thrombin (Sigma) and 5 mM CaCl₂. Then, matrices were transferred to Ultra-Low Cluster 24-well plate (Corning) containing 500 μl of buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, pH 7.4). Control wells that served as 100% released control contained only PDGF-BB and IL-1Ra variants in 500 μl of the buffer. Every 24 h, buffers were removed from wells and kept at −20 °C. Wells were replenished with fresh release buffer. For the 100% release control well, 20 μl of buffer was taken out every day and stored at −20 °C. After 7 days, the cumulative release of PDGF-BB and IL-1Ra variants was quantified by ELISA using the 100% released control as reference (PDGF-BB DuoSet, IL-1Ra/IL-1F3 DuoSet; R&D Systems). For release assays with plasmin, the same method was used except that the release buffer contained 100 μU/ml of plasmin (Roche).

ELISA plates (96-Well Medium Binding, Greiner bio-one) were coated with solutions of 100 nM of human plasma fibronectin (Sigma), human vitronectin (Peprotech), human tenascin C (R&D Systems) or human fibrinogen (Enzyme Research Laboratories) in PBS for 1 h at 37 °C. Wells were washed with washing buffer (PBS-T, PBS with 0.05% Tween-20) and blocked with 1% BSA in PBS-T for 1 h at room temperature. Then, wells were incubated with 100 nM of GST-fused PlGF fragments (in PBS-T with 0.1% BSA) for 1 h at room temperature. GST (100 nM) only was used as a negative control. Wells were washed three times with washing buffer and incubated with 0.1 μg/ml of HRP-conjugated antibody against GST (GE Healthcare, RPN1236V) in PBS-T with 0.1% BSA for 45 min at room temperature. Wells were then washed three times with PBS-T and detection was done with tetramethylbenzidine substrate and measurement of the absorbance at 450 nm.

ELISA plates (Medium binding, Greiner Bio-one) were coated with 100 nM of ECM proteins in 50 μl of PBS for 1 h at 37 °C; human plasma fibronectin (Sigma), human vitronectin (Peprotech), human tenascin C (R&D Systems), human fibrinogen (Enzyme Research Laboratories). Then, wells were washed with PBS-T (PBS with 0.05% Tween-20) and blocked with 300 μl PBS-T containing 1% BSA for 1 h at room temperature. ECM and control wells (no ECM and blocked with BSA) were further incubated 1 h at room temperature with solutions of murine PDGF-BB (Peprotech), IL-1Ra (R&D Systems) or PlGF_123–141-fused proteins at concentrations ranging from 0 to 100 nM (50 μl in PBS-T containing 0.1% BSA; PROKEEP tubes, Watson bio lab). Then, wells were washed three times with PBS-T and bound PDGF-BB and IL-1Ra variants were detected using biotinylated antibodies in PBS-T containing 0.1% BSA. Antibodies used were from PDGF-BB DuoSet ELISA (R&D Systems, DY8464) for PDGF-BB and IL-1Ra/IL-1F3 DuoSet ELISA (R&D Systems, DY480) for IL-1Ra. To calculate the dissociation constants (K_d) specific-binding values were fitted with a one site-specific binding model using A₄₅₀ nm = B_max*[growth factors or IL-1Ra variants]/(K_d + [growth factors or IL-1Ra variants]).