Calcein am

The viability of SkM on HAM and EF-HAM scaffolds was assessed through Live/Dead assay which distinguishes live from dead cells through staining with Calcein AM (Cayman Chemical, Ann Arbor, MI, USA) and ethidium homodimer-1 (EthD-1; Thermo Fisher), respectively. SkM were seeded on scaffolds at a 10,000 cells/cm² seeding density and cultured for 48 h before staining procedure. For staining, cells were incubated with a mix solution of 2 µM of Calcein AM and 4 µM of EthD-1 prepared in Hank’s 1X Balanced Salt Solutions (HBSS; Cytiva, Marlborough, MA, USA) at room temperature for 30 min. Staining solution was then replaced with HBSS and the images of stained cells were captured at five different fields using fluorescence microscope (Nikon, Tokyo, Japan) before the number of live and dead cells were counted and averaged.

The co-culture model and neuronal monoculture model at day 30 after mixing the cells were treated with 15 μg/mL L-Glutamate (Wako) for one week. To investigate cell viability, the cells were treated with 1 μg/mL Calcein-AM (DOJINDO) in the culture media for 1 h, washed with PBS, and fixed with 4% paraformaldehyde for 16 h at 4 ℃. Then, fluorescent signals of Calcein-AM were detected by In Cell Analyzer 6000 (Cytiva, Marlborough, United States). Calcein-positive and MAP2-positive neurons were counted by In Cell Developer (Cytiva, Marlborough, United States). The surviving neuron ratios were calculated as Calcein-positive and MAP2-positive neurons/Hoechst.