Of all protocols, the decellularization methods, which efficiently removed nuclear materials examined as detected by immunofluorescent staining, were quantitatively tested for sample DNA and GAG contents. The lenticules were lyophilized in a freeze dryer (FD5512, IlShin, Gyeonggi-do, Korea) and weighed. The DCLs were digested in 0.2 M sodium phosphate buffer (pH 6.4) containing 125 μg/mL papain (Sigma), 10 mM cysteine hydrochloride (Sigma), 0.1 M sodium acetate (Junsei, Tokyo, Japan), and 2 mM EDTA (Sigma) for 3 h at 65°C as described previously [25 (link)]. The DNA content was measured by using a DNA quantitation kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol. Briefly, 5 μL solution of dissolved lenticules (10 mg/mL) was added to 1 mL TEN buffer (100 mM Tris, 2 M NaCl, 10 mM EDTA, pH 7.4) containing Hoechst 33528 (1 μg/mL). Fluorescence intensity was read using a 460 nm emission filter with a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany). The DNA contents were calculated from a standard curve determined by using calf thymus DNA.
Sulfated GAG in the DCL was measured with a Blyscan™ glycosaminoglycan assay kit (Biocolor, County Antrim, UK). GAG quantification was performed according to the manufacturer's protocol. Absorbance was measured using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 656 nm.
Sulfated GAG in the DCL was measured with a Blyscan™ glycosaminoglycan assay kit (Biocolor, County Antrim, UK). GAG quantification was performed according to the manufacturer's protocol. Absorbance was measured using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 656 nm.