Mtt proliferation assay

To assess cell proliferation behavior, an MTT proliferation assay (Sigma Aldrich) was used with extracts from 3.6 wt% PEG-NHS-Fgn-R2-SNAP hydrogel composites and PEG-NHS-Fgn controls incubated for 24 hours in DMEM at culture conditions. The MTT Cell Proliferation Assay measures the cell proliferation rate, when changes in cellular metabolism lead to apoptosis or necrosis, this results in a reduction in cell viability. L929 fibroblasts were seeded with conditioned media containing 100uL of extracts in 96-well plates at a density of 1× 10⁴ cells/well. Cells cultured in DMEM served as controls. After 24 hours, the media from each well was replaced with 50uL of MTT solution (1 mg/mL in PBS) and plates were incubated for an additional 2 hours. The MTT solution was exchanged for 100uL isopropanol/well and the absorbance of each well was measured at 570 nm (reference 650 nm) using a Synergy HT Multi-Mode Microplate Reader (BioTek, USA). The relative cell viability was determined as follows (Equation 5):
$$\%cellviability=\frac{{\text{ Abs}}_{\text{hydrogel}}}{{\text{ Abs}}_{\text{control}}}\times 100$$
where Abs_hydrogel and Abs_control are the absorbance for cells cultured in hydrogel media extracts and standard media, respectively. Four independent cultures were prepared for hydrogel composites and control hydrogels. Samples with relative cell viabilities greater than 70% were considered non-cytotoxic.