Synergy 2 luminescent plate reader

To assess the ability to release growth factors, 50 uL HS (250 mM NaCl) and PS (145 mM NaCl) gels (10 mg/mL fibrinogen) were fabricated with 100 ng/mL FGF-2 (Peprotech, 100-18b) and 100 ng/mL VEGF-165 (Shenandoah Biotechnology, 100–44) in the bottom of 1.6 mL Eppendorf tubes. To fabricate these gels, a PBS solution of 400 ng/mL FGF-2 and 400 ng/mL VEGF-165 was used instead of the normal 4X cell solution in Step 3 of Section 2.1 above. After gelation, 1 mL of 0.5% protease-free BSA (Sigma, A3059) in PBS was added to each tube and was completely replaced daily. After 0, 1, and 7 days, the supernatant was removed and 50 uL of 0.2 mg/mL human plasmin (Enzyme Research Laboratories) in HEPES buffer (pH 8.5, Boston Bioproducts) was added. Fibrin degradation occurred overnight at 37°C. Growth factor retention was measured using ELISAs (Peprotech, 900-K08 and 900-K10) and a BioTek Synergy 2 luminescent plate reader (Gen5 software).

Cell survival assay and mitochondrial membrane potential assays were performed using the Cell Titer‐Glo Luminescent Cell Viability Assay kit (Promega, Wisconsin) and Mitochondrial membrane potential assay kit with JC‐1 (Beyotime, China). All the analyses were performed according to the manufacturer's instructions. Luminescence and fluorescence were recorded with a Biotek Synergy2 Luminescent plate reader.