Celltiter glo luminescence cell viability assay kit

All solvents were of analytical grade (>95% purity). All buffer components were of biological grade and used without further purification. Iridium chloride hydrate, ppy, dfppy, H₂dcbpy and NH₄PF₆ were purchased from Alfa Aesar. Cisplatin, rapamycin, DMSO, PLE, PI, NAC, MTT, H₂DCFDA, JC-1, 3-MA, CCCP, CQ, AO and Annexin V-FITC apoptosis detection kit were purchased from Sigma-Aldrich. MTDR was purchased from Invitrogen. Lipofectamine^® 3000 was purchased from Thermo Fisher Scientific. Hoechst 33342 and z-VAD-fmk were purchased from Beyotime. Caspase-Glo^® 3/7 assay kit and CellTiter-Glo^® luminescence cell viability assay kit were purchased from Promega.
Ligands L₁ (dimethyl 2,2′-bipyridine-4,4′-dicarboxylate), L₂ (diethyl 2,2′-bipyridine-4,4′-dicarboxylate), L₃ (dibutyl 2,2′-bipyridine-4,4′-dicarboxylate) and L₄ (diisobutyl 2,2′-bipyridine-4,4′-dicarboxylate) were synthesized according to reported procedures24 . The cyclometalated Ir(III) chloro-bridged dimers [Ir(ppy)₂Cl]₂ and [Ir(dfppy)₂Cl]₂ were prepared according to literature methods57 58 (link). The purity of synthesized complexes was analysed via combustion analysis and was found to be ≥ 95% pure. All the tested complexes were dissolved in DMSO just before the experiments, and the final concentration of DMSO was 1% (v/v).

siRNA-treated U2OS cells were seeded on 96-well plates and treated with increasing doses of mitomycin C (MMC; Sigma) the following day. Cell viability was determined using the Cell Titer-Glo Luminescence Cell Viability Assay kit (Promega) and Spectramax M5 (Molecular Devices) 6 days following continued drug treatment.

To measure the viability of CSC and CSCMKO cells treated with HG or NG in the presence or absence of JNK inhibitor, 5 × 10³ cells were seeded in 96-well plate in complete medium in cell culture incubator at 37 °C with 5% CO₂. After 24 h of incubation, cells were treated with HG or NG as well as HG with JNK inhibitor or vehicle control. We also measured the viability of cells treated with/without H₂O₂. We pretreated the cells with 5 µM of final concentration of JNK inhibitor for 30 min before treating them with HG, NG, or H₂O₂. Cells were treated with HG, NG, or H₂O₂ for 24 h. At the end of treatment, cell viability was determined using CellTiter-Glo Luminescence Cell viability Assay kit (Promega, Cat #G7571) by measuring luminescence (ATP level) as per assay protocol. Medium without cells were used as background luminescence. Values were presented as a RLU.