The lyophilized matrix was cut into 10 mg pieces then were rinsed in 75% ethanol and sterile PBS. 60 μl ADSCs suspension (10 (Keane et al., 2015 (link)) cells) were seeded in the DAM and ADM and placed into 24-well transwell culture inserts in a 0.4 μm layer. After 24 h in culture, the ADSC-matrix composites were transferred to a new 24-well plate for better exposure to the medium.
Cell viability and adhesion rate were determined using the PrestoBlue™ Cell Viability Reagent (Thermo, United States). An equal number of cells were seeded in blank wells as the control group in the 24-well plate (n = 3 for each group). PrestoBlue solution (400 μl) was added to each well, and the plates were incubated at 37°C for 30 min. After incubation, the fluorescence intensity with excitation wavelength at 560 nm and emission wavelength at 590 nm was measured using a microplate reader (Multiskan, Thermo Fisher Scientific, United States). The LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Thermo Fisher) was used to visualize the attachment and stretching of cells at days 2 and 5 after cell seeding. Confocal microscopy was performed using a Leica TCS SPII microscope (Leica, Allendale, NJ).
Cell viability and adhesion rate were determined using the PrestoBlue™ Cell Viability Reagent (Thermo, United States). An equal number of cells were seeded in blank wells as the control group in the 24-well plate (n = 3 for each group). PrestoBlue solution (400 μl) was added to each well, and the plates were incubated at 37°C for 30 min. After incubation, the fluorescence intensity with excitation wavelength at 560 nm and emission wavelength at 590 nm was measured using a microplate reader (Multiskan, Thermo Fisher Scientific, United States). The LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Thermo Fisher) was used to visualize the attachment and stretching of cells at days 2 and 5 after cell seeding. Confocal microscopy was performed using a Leica TCS SPII microscope (Leica, Allendale, NJ).