Luminescence reader

The cell-surface expression levels of wild-type KOR and its mutants were measured using an enzyme-linked immunosorbent assay (ELISA). In brief, HEK293T (ATCC CRL-11268) cells were transiently transfected with wild-type KOR and KOR mutant DNA at the same quantity. After 24 h, cells were plated in poly-l-lysine-coated 96-well white clear-bottom plates in plating medium (DMEM + 1% dialysed FBS) at a density of 40,000–50,000 cells in 200 μl per well and incubated overnight. The next day, plates were decanted and fixed with 4% (w/v) paraformaldehyde for 10 min at room temperature. Cells were then washed twice with 1× phosphate-buffered saline (PBS) (pH 7.4) and blocked by 1× PBS containing 0.5% (w/v) non-fat milk for at least 30 min at room temperature followed by incubation with anti-Flag (M2)–horseradish peroxidase-conjugated antibodies (Sigma-Aldrich, A8592) diluted 1:20,000 in the same buffer for 1 h at room temperature. After washing three times with 1× PBS, 1-Step Ultra-TMB ELISA substrate (Thermo Fisher Scientific, 34028) was added to the plates and the plates were incubated at 37 °C for 15–30 min and terminated by addition of 1 M sulfuric acid (H₂SO₄) stop solution. Finally, the plates were read at a wavelength of 450 nm using the BioTek Luminescence reader. The data were analysed using GraphPad Prism (v.9.3.1).

The ROS levels in the rat retina were analyzed using luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, Sigma) and lucigenin (bis-N-methylacridiniumnitrate, Sigma). Briefly, retinas were isolated at weeks 1, 2, and 3 after surgery and then homogenized with 10 mM PBS. Tissue samples were transferred into vials in the presence of PBS-HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). The levels of ROS were assessed by adding the chemilumigenic probes lucigenin and luminol (final concentration of 0.2 mM). The luminescent counts were recorded at 1 min intervals at room temperature via luminescence reader (BioTek). Results were given as counts per min (counts/min) for a counting period of 5 min (rlu/mg tissue).

Ascites‐derived spheroids from five different high‐grade serous ovarian cancer patients were enriched with 15 µm cell strainers as described before. Approximately 1 × 10⁴ counted cells were seeded in each well of a 96‐well plate resuspended in MCDB medium (90 µL/per well). Then, a total of 10 µL MCDB medium containing metformin (Merck KGaA; Darmstadt, Germany), cisplatin (Pharmacy, University clinic Hamburg‐Eppendorf, Germany), IACS010759 (Selleck Chemicals, #S8731), metformin + cisplatin or IACS 010759 + cisplatin were added to each well to reach following final concentrations: 0, 5 mm metformin, 0, 3.3, 33.3 µm cisplatin and 50 nm IACS 010759. Each treatment condition was plated in triplicates. After 48 h, 80 µL volume of spheroids from each well was mixed with equal amount of CellTiter‐Glo® Luminescent Cell Viability Assay Chemistry (Promega; Madison, Wisconsin, USA) in a white 96 Well Polystyrene Microplate (Greiner; Bio‐one, Kremsmünster, Austria) and shacked for 10 min. After 20 min further incubate at room temperature, the cell viability was measured using a luminescence reader (BioTek; Winooski, VT, USA).