Nunclon sphera microplate

At 24 h post transfection, cells were reseeded with complete medium in 24-well plates without adhesion (Nunclon™ Sphera™ Microplates, Thermo Fisher Scientific). Sphere formation was evaluated on day 6 at ×10 magnification with a phase-contrast inverted microscope.

hiPSCs were treated with TrypLE Express (Thermo Fisher Scientific) and transferred onto Nunclon Sphera Microplates (Thermo Fisher Scientific) in Primate ES cell culture medium supplemented with 10 µM Y27632 (Wako). Cells were cultured for 3 to 5 days to allow EB formation. EBs were attached to a gelatin-coated plate and cultured in DMEM supplemented with 10% FBS for an additional 10 to 12 days.

Proliferation curves were generated using an IncuCyte ZOOM system (Essen BioScience) on cells seeded on microplates (TPP or Nunc Edge) based on phase contrast images taken at 2 h intervals for the duration of the experiments, except for Supplementary figure S16 where plates were scanned once per day. Spheroid growth assays were performed in Nunclon™ Sphera™ Microplates (Thermo Fisher Scientific). Cells were seeded at a density of 8000 cells per well (except for M202, M207 and M257 cells which were seeded at 32,000 cells per well) in 96 well plates or 150 cells per spheroid.

The in vitro antitumor efficacy of IPI549@HMP-sensitized radiotherapy was evaluated using Calcein-AM/PI Double Staining Kit (Dojindo). Briefly, CT26 cells were cultured in Nunclon Sphera Microplates (Thermo, Catalog No.174925) to obtain multicellular spheroids (MCSs). The formed MCSs were co-incubated with PBS/HMP/IPI549@HMP for 12 h followed by X-ray radiation (225 KV and 8 mA for total 6 Gy) based on groups. After further incubation for 24 h, the MCSs were stained and observed under confocal microscope to estimate cytotoxicity.

The human hepatic cell line HepaRG was cultured in a growth medium consisting of William's Medium E (10% fetal bovine serum, 1% 100 U/mL penicillin, 1% 100 μg/mL streptomycin, and 50 μM hydrocortisone hemisuccinate), then cultured in a cell incubator at 37°C and 5% CO₂. For toxicity studies (cell viability and PA induction studies), HepaRG cells were seeded in Nunclon™ Sphera™ Microplates (Thermo Fisher Scientific; 9000 cells per well in 100 μL). The process of cell differentiation is described previous literature [23 (link)]. Before toxicity studies, differentiated HepaRG cells were incubated in assay medium (growth medium containing 2% FBS) supplemented with 0.5% DMSO. For all assays, PAs were dissolved in PBS (0.05 M, pH 7.4) to make a stock solution of 100 mM. At this stage, the cells were ready to be used for toxicity studies. Cells that were not immediately used were kept in a differentiation medium for a maximum of three additional weeks. The medium was refreshed every 1-2 days during culturing.