Picogreen dna quantification assay

Different cylindrical scaffolds (SF, S-ELP and S-ELP-DHT) with a diameter of 5 mm and a height of 3 mm were first prepared and placed in a 24-well plate. BMSCs or chondrocytes were seeded onto the top surface of each scaffold at a density of 2 × 10⁷ cells/mL (200 μL suspension, a total of 4 × 10⁶ cells), and then cultured in an incubator to ensure adequate cell adhesion to scaffolds. After culture for 6, 12 and 24 h, the samples were removed from culture medium, rinsed twice with PBS, fixed with 2.5% glutaraldehyde at 4 °C for 30 min and finally dehydrated with increasing concentrations of ethanol solutions (30–100%). Scaffolds were then air-dried, sputtered with gold-palladium, and analyzed with SEM. Next, the number of cells adhered onto the scaffolds was evaluated using PicoGreen® DNA quantification assay (Thermo Fisher Scientific) after removal of loosely adherent or unbound cells by washing with PBS. Cell seeding efficiency was calculated using the following equation: Cell seeding efficiency (%) = (Total DNA content of cells in each scaffold)/Total DNA content of initially seeded cells × 100% [35 (link)].

Our DNA corona formation method has been described previously (37 (link)). In brief, DNA coronas were formed by incubating NPs (500 pM) with E. coli DNA (0.25 mg/mL, #J14380, Fisher Scientific, Hampton, NH), in Dulbecco’s Phosphate Buffered Saline (PBS; pH 7.3-7.5, #28374, Thermo Fisher). The DNA and NPs were mixed by pipetting up and down and then incubated at room temperature (RT) for 45 min. Then, 1 µL of 1M hydrochloric acid was added to the 2 µm NPs when incubating with DNA to protonate the amine functional groups. Unbound DNA was removed by centrifugation (18,000 rcf) and resuspension in PBS (×3). Removal of unbound DNA using this washing process was confirmed previously (37 (link)). The resulting NPs with a DNA corona were characterized with DLS and TEM in the same way as the bare NPs, as described above. The concentration of DNA in the corona was measured using the PicoGreen DNA quantification assay (#P7589, Thermo Fisher), according to the manufacturer’s instructions. Fluorescence intensity (Ex: 480 nm, Em: 520 nm) was measured with a plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA). The background signal from the bare NPs was subtracted from the overall signal.