Calcein am propidium iodide

The effects of dexamethasone on MSCs were quantified by Calcein‐AM/propidium iodide (PI) (Dojindo). The samples were treated with different concentrations of dexamethasone for 3 days; then the cells were rinsed with PBS three times and incubated in PBS containing 2 μmol·L⁻¹ Calcein‐AM and 4 μmol·L⁻¹ PI in the dark for 15 min. The cells were then detected by a fluorescence microscope (Olympus, Tokyo, Japan) in the dark. Live cells showed green fluorescence, whereas the dead cells showed red fluorescence.

The viability of NSCs and OLGs embedded in sodium alginate/gelatin hydrogels after extrusion was investigated by staining with Calcein-AM/propidium iodide (PI) (Dojindo, Japan). The 3D bioprinting scaffolds were seeded in 12-well culture plate (Corning) with complete DMEM/F12 medium and the medium was changed every other day. Cultured for 3 and 5 days, the scaffolds were performed cell viability test at Days 3 and 5, respectively. Three-dimensional bioprinting scaffolds were washed with PBS 2 times and then were immersed in 100 μl staining solution (PI at 4.5 μM and Calcein-AM at 2 μM) for 30 min at 37°C in dark. Then, the scaffolds were washed with PBS two times. Leica DMi8 Confocal Microscope (Leica Microsystems, Wetzlar, Germany) was used to take images of samples. Each image was obtained by z-axis superposition. We randomly selected six visual fields of each sample (n = 3) at 200× magnifications for viability statistics.
Cell viability = the number of living cells (green, living cells)/(number of living cells + number of dead cells (red, living cells)) × 100%

γ-GC with a purity of over 95% (CAS no. 636-58-8) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies against 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), TNF receptor-associated factor 2 (TRAF2), eukaryotic initiation factor 2 (eIF2α), phosphorylated eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), c-Jun NH 2-terminal kinase (JNK), and phosphorylated JNK (p-JNK) were obtained from Cell Signaling Technology (Massachusetts, USA). Antibodies against Bax, Bcl-2, and GAPDH were purchased from Bioworld Technology (St. Louis, Missouri, USA). Antibodies against caspase-3 were purchased from Proteintech Group (Wuhan, China). An antibody against p-PERK was obtained from Signalway Antibody LLC (Maryland, USA), and an antibody against p-IRE1α was obtained from Affinity Biosciences (Jiangsu, China). Cell Counting Kit-8 (CCK-8) and calcein-AM/propidium iodide (PI) were purchased from Dojindo Molecular Technologies (Tokyo, Japan). An Annexin V-PE/7-AAD apoptosis detection kit and TUNEL kit were obtained from Vazyme Biotech Co. (Nanjing, China). ROS, malondialdehyde (MDA), and GSH assay kits were obtained from Beyotime Biotechnology (Shanghai, China).