Live dead staining solution

To assess cell viability by live/dead staining, cell-loaded SF scaffolds and ECM-modified scaffolds were incubated with 0.2 ml live/dead staining solution (Invitrogen) for 30 min [23 (link)]. After washing with PBS, the samples were examined under a confocal scanning microscope (Nikon A1) to distinguish live and dead cells.

The prepared BAM (Group C) was immersed in α-MEM at 37°C for 72 h, and the extract was collected for later use. The third passage ASCs were seeded in 96-well plates at a concentration of 5000 cells/well and incubated in α-MEM (containing 10% FBS) at 37°C with 5% humidified CO₂ for 24 h. The culture medium was changed by BAM extract and control α-MEM respectively. At 1, 3, 5, 7 days, the culture medium was replaced by fresh α-MEM, ASCs were incubated with 10 μl of Cell Counting Kit-8 (CCK-8, Invitrogen, Carlsbad, CA, United States) reagent in 5% humidified CO₂ for 2 h at 37°C. The absorbance at 450 nm was measured by a microplate reader. At the same time, live/dead staining was used to assess the ASCs viability in the BAM extract. At 3, 5, 7 days, the culture medium was aspirated, the ASCs were washed once with PBS and then incubated with 100 μl live/dead staining solution (Invitrogen) at 37°C with 5% humidified CO₂ for 15 min, as recommended by the manufacturer. After washing with PBS, the ASCs were examined by Nikon Eclipse Ti2-U fluorescence microscope to distinguish the live and dead cells.

The alamarBlue assay (Thermo Fisher Scientific, Brisbane, Australia) was used to quantitatively assess the cell metabolism following the manufacturer protocol. Briefly, at 1 d, 7 d and 14 d timepoints, the MSC medium was removed from 4 samples of each mesh group, and the samples were rinsed with PBS and transferred to a fresh 48-well plate. The samples were then incubated with 330 µL of fresh culture medium containing 10% (v/v) of alamarBlue solution for 4 h at 37 °C with 5% CO₂. After the incubation, 3 aliquots of 100 µL from each sample medium were transferred to black-wall 96-well plates. The fluorescence was read at 545/590 nm (excitation/emission) with a CLARIOstar microplate reader (BMG Labtech, Mornington, Australia).
LIVE/DEAD assay was used to show the distribution of live and dead cells attached on the mesh samples. Briefly, after 1 d and 7 d of cell seeding, the mesh samples were moved to a fresh 48-well plate and washed twice with PBS solution. The disk samples were incubated for 30 min in 300 µL of LIVE/DEAD staining solution (Thermo Fisher Scientific, Brisbane, Australia) containing 2 µM calcein and 4 µM ethidium. The stained samples were imaged with a fluorescent microscope (Zeiss Axio Observer 7, Carl Zeiss, Oberkochen, Germany) immediately after the staining.