Nunclon sphera microplates 96u well plate

SFEBq-based differentiation was performed as previously described [19 (link), 20 (link)]. iPSCs were dissociated into single cells and quickly re-aggregated in DFK 5% medium (DMEM/F-12 Ham medium (Sigma-Aldrich) containing KnockOut™ Serum Replacement (Thermo Fisher Scientific), NEAA (Thermo Fisher Scientific), 2-mercaptoethanol (Nacalai), GlutaMAX (Thermo Fisher Scientific), SB-431542, Dorsomorphin (Tocris, Minneapolis, MN, USA), and Y-27632 (Tocris)) (9000 cells/well) using a Nunclon Sphera Microplates 96U-Well Plate (Thermo Fisher Scientific). The medium was changed every 4 days until day 12, after that we changed to neurobasal medium supplemented with B27 supplement (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific) for further neural differentiation. Neurospheres were fixed in 4% paraformaldehyde for 30 min at room temperature followed by dehydration with 30% sucrose overnight and then cryosectioned at 20 μm with Leica CM1860 Cryostat (Leica Biosystems, Wetzlar, Germany).

Retrorsine (RTS), monocrotaline (MCT), retronecine (RN), platyphylline (PLA), and camptothecin (CPT) were purchased from J&K Scientific Ltd. (Beijing, China). 0.25% Trypsin/1 mM EDTA, 0.25% Trypsin, William's E Medium (no phenol red), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS, Australia Origin), Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 and Propidium Iodide (PI), Tali™ Cell Cycle Kit, the fluorescent probes Hoechst 33342, Mito Tracker Red CMXRos, ER-Tracker™ Green (BODIPY™ FL Glibenclamide) for live-cell imaging, BODIPY™, Fluo-4 direct calcium reagent, Total Reactive Oxygen Species (ROS) Assay Kit 520 nm, and Nunclon™ Sphera™ Microplates (96 U-Well Plate) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Cell Counting Kit-8 and Mitochondrial Membrane Potential Assay Kit with JC-1 were purchased from Beyotime (Nanjing, China). Hydrocortisone-hemisuccinate (sodium salt) and dimethyl sulfoxide (DMSO) were acquired from Solarbio Science﹠Technology Co., Ltd. (Beijing, China). The HepaRG cell line was purchased from Millipore (BeNa Culture Collection, China).