Proteinase k of 0.5 ml

LIVE/DEAD® staining (Cell viability, Invitrogen – using Olympus fluorescence microscope) was performed at 7 and 14 days to assess cell viability. Total DNA content was used to determine the cell count of each as previously described [14 (link)]. Proteinase K of 0.5 mL (Sigma-Aldrich) at a concentration of 0.5 mg/mL was added to each well, and plates were incubated at 56°C overnight for enzymatic lysis of cells and DNA release. Aliquots (50 mL) were mixed with equal volumes of 0.1 g/mL PicoGreen® dye solution (Invitrogen) in 96-well plates. Samples were then excited at 480 nm with an emission wave length of 520 nm using a plate reader (SpectraMax M5). Total DNA concentration was compared to a standard curve generated from serial dilutions of CD34⁺/CD31⁻ cells to calculate the number of the cells in each well. Samples prepared without cells served as blank controls.

hASCs cell sheets were transferred into 24-well plates and maintained in GM for 21 days. LIVE/DEAD^® staining (Cell viability^®, Invitrogen – using a Lumar System) was performed at 1, 7, 14, and 21 days to assess hASC viability in cell sheets.
Total DNA content was used to determine the cell count of each cell sheet as previously described.^{19 (link)} Proteinase K of 0.5 mL (Sigma-Aldrich) at a concentration of 0.5 mg/mL was added to each well, and plates were incubated at 56°C overnight for enzymatic lysis of cells and DNA release. Aliquots (50 μL) were mixed with equal volumes of 0.1 g/mL PicoGreen dye solution (Invitrogen) in 96-well plates. Samples were then excited at 480 nm with an emission wavelength of 520 nm using a plate reader (Wallac 1420 Multilabel HTS Counter). Total DNA concentration was compared to a standard curve generated from serial dilutions of hASCs to calculate the number of the cells in each well. Samples prepared without cells served as blank controls.