Echorevolve fluorescence microscope

The live–dead analysis was performed after 24 h, 72 h, and 168 h of incubation using the Live/Dead Cell Staining Kit from Enzo Life Sciences (Lausen, Switzerland) following the manufacturer’s guidelines. Briefly, after washing the cells with 1 × PBS, 150 µL of staining solution containing calcein-AM and propidium iodide for staining live and dead cells, respectively, was added per well followed by incubation for 15 min at 37 °C, 5% CO₂, and 95% humidity. Afterwards, the fluorescence staining was directly visualized using the EchoRevolve fluorescence microscope (Echo, San Diego, CA, USA), distinguishing between calcein-AM (green dye, Ex/Em = 488/515 nm) and propidium iodide (Ex/Em = 570/602 nm).

The focal adhesions were stained after 24 h, 72 h, and 168 h of incubation for hG-MSCs using the Actin Cytoskeleton/Focal Adhesion Staining Kit from Merck (Darmstadt, Germany), according to the manufacturer’s instructions. In brief, after fixing and permeabilizing the cells with 4% paraformaldehyde and 0.1% Trition X-100 in 1 × PBS, respectively, the hG-MSCs were blocked by adding 1% BSA in 1 × PBS. The actin filaments were stained with TRITC-conjugated anti-phalloidin (Ex/Em 540/565 nM) for focal adhesion with the anti-vinculin monoclonal antibody (purified clone 7F9) followed by the FITC-conjugated secondary antibody (Ex/Em 495/520 nm). This was followed by nuclei counterstaining with 4′,6-diamidin-2-phenylindol (DAPI, Ex/Em 358/461 nm). Brightfield and fluorescence pictures were taken using the EchoRevolve fluorescence microscope (Echo, San Diego, CA, USA).