Cell mito stress test assay and Glycolysis stress test assay

Mitochondrial respiration experiments were conducted using the Mito Stress Test Kit and Glycolysis Stress Test Kit (Seahorse Bioscience, Billerica, MA, USA). OCR and ECAR were measured using a Seahorse the XF24 analyzer (Seahorse Bioscience, Billerica, MA, USA).

Cell mito stress test assay was performed as previously described [31]. Cells were seeded into 96-well cartridges and pre-equilibrated with the assay medium for 1 h. OCR was assayed under basal conditions followed by sequentially loading pre-warmed oligomycin, FCCP, rotenone & antimycin A into the sensor cartridge. ATP coupler oligomycin allows for measurement of oxygen consumption for ATP synthesis. Uncoupling FCCP reagent is used to measure maximal OCR level for evaluation of the spare respiratory capacity. Rotenone and antimycin A arrests mitochondrial respiration by prohibiting mitochondrial complexes I and II. Final concentrations of these reagents have been demonstrated in a previous study by Tan et al. [13].

Glycolysis stress test assay was carried out following the vendor instructions (Seahorse Bioscience, Billerica, MA, USA). Briefly, cells were calibrated by the assay medium. Glucose, oligomycin and 2-deoxyglucose (2-DG) were then sequentially added in the assay medium. ECAR was detected under basal conditions and after separate treatments of glucose, oligomycin, and 2-DG. Glucose addition promotes glycolysis, oligomycin treatment suppresses oxidative phosphorylation and permits analysis of maximal cellular glycolytic capacity, and 2-DG treatment is used to inhibit glycolysis.