Uv vis synergy h1 plate reader

The amount of ssDNA in the NACC self-assembled fibers for different volume fractions of type I collagen and ssDNA oligomer was assessed fluorometrically by measuring the intensity of a fluorescent DNA stain. Solutions of different volume fractions were diluted at a 1:2 volume ratio of fiber solution to SYBR Safe DNA stain (Invitrogen). The stain concentrate was diluted first at a 1:10,000 volume ratio in deionized water. Then the mixtures were incubated protected from light for 30 minutes at room-temperature. After incubation, the mixtures were centrifuged at 2000g for five minutes. Supernatant was plated in duplicate into black walled 96 well microplates and fluorescence intensity was measured by exciting at 488 nm and detecting at 520 nm using a UV-Vis Synergy H1 plate reader (BioTek). ssDNA in the supernatant was quantified using a standard curve fit with a 4-parameter logistic regression to correlate fluorescent intensity to ssDNA concentration. Bound ssDNA for the different volume fractions was calculated as the difference of the initial amount of ssDNA in the solution and the amount of ssDNA measured in the supernatant.

Cell proliferation was determined using a cell proliferation stain. Prior to seeding the hydrogels, cells were fluorescently stained with Cytopainter Orange (Abcam, Cambridge, UK), which stains live cells orange at an excitation wavelength of 488 nm and emission wavelength of 542 nm and is incorporated into dividing cells. Cytopainter was reconstituted according to the manufacturer’s instructions. The staining solution was prepared by diluting 1 μL of stain with 500 μL of sterile PBS. HUVECs were trypsinized, pelleted, and resuspended in the staining solution. The cells were incubated in the solution for 30 minutes in a cell culture incubator. The HUVECs were then repelleted, the staining solution removed and rinsed once with PBS, and resuspended in HGM. Then these cells were seeded at 25,000 cells/well onto hydrogels prepared in a black wall, clear bottom 96-well plate. Fluorescent intensity was measured at 2 days, 4 days, and 6 days with a UV–Vis Synergy H1 plate reader (BioTek, Winooski, VT, USA). HGM blanks were used to subtract any background fluorescence due to the cell culture media. Standard curves were prepared by measuring the fluorescence of known numbers of stained cells.