Live dead cell assay kit

Incubated spheroids were transferred on to 96-well cell plate and cultured for 2 h in a medium containing Doxorubicin hydrochloride (Dox-HCl, Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 2, 4, 6, 8, and 10 μg/mL. After Dox treatment, the spheroids were stained to measure viability using Live/Dead Cell Assay kit (Sigma-Aldrich, Bayswater, Australia) with staining for 20 min at 37 °C. The stained spheroids were imaged under the fluorescence microscope (IX73, Olympus, Japan) and the Live/Dead staining results were evaluated using Image J by the threshold function.

Hydroquinone (HQ), sodium carbonate (Na₂CO₃), phosphate-buffered saline (PBS), penicillin-streptomycin, trypsin-EDTA, a Live/Dead Cell assay kit, and MTT-assay kit were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO, USA). Further, 4,4′-Difluorobenzophenone (4,4′-DFBP) and diphenyl sulfone (DPS) were purchased from TCI Co. Ltd. (Tokyo, Japan). Fetal bovine serum and Dulbecco’s modified Eagle medium (DMEM) were purchased from GE Healthcare Life Sciences (Logan, UT, USA). 4,4′-(1,4Phenylenebis(oxy)) diphenol (PD) was kindly provided by the Organosynthesis Lab., Dept. of Chemistry of Incheon National University (Incheon, Korea). The ALP assay kit was purchased from AnaSpec. Co. Inc. (Fremont, CA, USA). L-929 mouse fibroblast cells were obtained from the Korean Cell Line Bank Co. (Seoul, Korea).
P(E2–E4)K powder, graphene oxide powder (purity: >99 wt %, lateral dimension: ≥7 μm, thickness: 1.1~1.3 nm, LS-Chem, Ochang, Korea), and milled carbon fiber (PX 35 Milled Carbon Fibers, average length: 150 μm, average diameter: 7.2 μm, specific gravity: 1.81 g/cm³, Zoltek Co., St. Louis, MO, USA) were used for the preparation of composite samples using synthesized PEEK copolymers.

HepG2 cells are seeded within a microwell array in a gut-liver axis chip and incubated in an incubator for 5 or 10 days to produce uniform-sized 3D spheroids. After the generation of spheroids, the spheroids were stained to measure viability using Live/Dead Cell Assay kit (Sigma-Aldrich, Bayswater, Australia) with staining for 20 min at 37 °C. Calcein-AM can permeate the plasma membrane and is hydrolyzed by the cytoplasmic esterase to form Calcein-AM that can emit the fluorescence. Propidium iodide (PI) is a nucleic acid-staining dye and it cannot permeate the plasma membrane. The stained hepG2 spheroids were imaged under a fluorescence microscope (IX73, Olympus, Japan).