Ldh release kit

Human colorectal adenocarcinoma cells (HT-29, ATCC number HTB-38, Wesel, Germany) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Bleiswijk, The Netherlands) with 10 % FCS (Gibco) and penicillin–streptomycin (Gibco). Colourless DMEM (Gibco) with 1 % FCS (Gibco) was used as assay medium. HT-29 cells were seeded at a density of 2.0 × 10⁴ cells/well in a Costar flat-bottom 96-well plate (Corning, Amsterdam, The Netherlands) and incubated overnight. Serial dilutions of GS and derivatives were added and incubated for 4 h at 37 °C. Plates were centrifuged for 10 min at 1,000 g, and the amount of LDH in the supernatants was determined (LDH release kit, Roche, Woerden, The Netherlands) following the protocol. Cytotoxicity testing was independently repeated three times.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) assays were used to investigate the toxicity of RUT against cells. MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA), and an LDH release kit was purchased from Roche Applied Science (Indianapolis, IN, USA). Cells were seeded in 48-well plates (5 × 10⁴ cells/well) and incubated at 37 °C for 24 h. Cells were treated with 1–20 μM RUT, and the plates were incubated at 37 °C for 24 h. A BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) was used to determine the absorbance at 550 nm (MTT) and 490 nm (LDH). Calculations of cell viability (%) and cytotoxicity (%) were based on the absorbance of RUT-treated cells relative to the absorbance of vehicle (DMSO 0.1%)-treated cells.