Mitochondrial respiratory capacity assays
Tumor cells from both 2D and 3D MTSs were digested to prepare single
cell suspensions. Oxygraph-2k mitochondrial function analyzer (Oroboros,
Innsbruck, Austria) was used to measure the cellular oxygen consumption
rate (OCR) under the following four conditions: Routine, normal cell
respiration level; Leak, ATP synthase was inhibited by 1 μL 4 mg/ml
oligomycin (Sigma-Aldrich, San
Luis, MO, USA); Electron transfer system (ETS): the oxygen consumption
level reached the maximum, with supplement of 1 μL 1 mM FCCP
(Carbonylcyanide p-trifluoromethoxyphenylhydrazone, Abcam, Cambridge,
UK) to destroy the proton gradient and mitochondrial membrane potential;
Residual oxygen consumption (ROX): the residual intracellular oxygen
consumption was measured by adding1 μL 1mM rotenone (Abcam, Cambridge,
UK) and 1 μL 1mM antimycin A (Abcam, Cambridge, UK) to inhibit
mitochondrial complex I/III and mitochondrial respiration. Basal
respiration: the OCR difference value between Routine and ROX; ATP
production: the OCR difference value between Routine and Leak; Maximal
respiration: the OCR difference value between ETS and ROX; Spare
respiratory capacity: the OCR difference value between ETS and Routine;
Non-mitochondrial respiration: the OCR value of ROX.