Nunclon sphera dishes

Cells were seeded in 60-mm non-adherent dishes (Nunclon Sphera Dishes, Thermo Fisher Scientific) at a density of 20,000 cells/mL and cultured to form cell aggregates. After 10 days of culturing, the medium containing the cells was transferred into a 15-mL conical tube, and the cells were allowed to settle (pellet) by gravity sedimentation for 5 min to separate cell aggregates from cells that remained in suspension. The supernatant was recovered by careful aspiration, and the number (N1) of cells in the supernatant was counted. The pellets were dissociated into single cells by incubation with trypsin/EDTA followed by pipetting, and the number (N2) of cells in the pellets was counted. The percentage of aggregation was calculated as follows: % aggregation = [number of aggregated cells (N2)/(number of aggregated cells (N2) + number of singlet cells (N1)] × 100, using data from three independent experiments.

22RV1 cells purchased from ATCC were routinely cultured in RPMI1640 medium (Gibco, Grand Island, NY) supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA). To develop olaparib resistant cell line, cells were maintained in the medium supplemented with 10μM of olaparib (Selleck Chemicals, Houston, TX) for 3 months until the viability reached over 95%.
293T cell line (Lenti-X 293T Cell Line) (34 (link)) was ordered from TaKaRa (Cat. No. 632180) cultured by DMEM medium supplemented with 10% Fetal Bovine Serum (FBS). All the cells were cultured at 37°C in 5% CO₂ incubator.
For 3D culture, we used Nunclon™ Sphera™ Dishes (Thermo Scientific, Cat. No. 174945) and added methylcellulose (0.5%; Fisher, Cat No. M-352) in RPMI 1640 growth medium supplemented 10% FBS to prevent excessive aggregation of cells in spheroid culture and to maintain even spheroid size.