Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR)

The mitochondrial respiratory activity and glycolysis status of live cells were measured by detection of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), using a Seahorse XF24 (Agilent, Santa Clara, CA, USA). Briefly, 3 x 10⁴ MCF-7 or MDA-MB-231 cells per well were plated on the XF24 culture plate and incubated at 37°C in 5% CO₂. The following day, the cells were incubated 1 h at 37 °C without CO₂ and washed 3 times with seahorse medium containing phenol red-free DMEM base (D5030, Sigma-Aldrich), 2 mM L-glutamine, and 1 mM pyruvate. The XF24 culture plate plus the cartridge pre-incubated with Seahorse XF Calibrant Solution (103059-000, Agilent) were mounted in the analyzer. OCR was recorded as pmolO₂/min, and ECAR was recorded as mpH/min. On the course of the assay, four sequential injections were performed after three readings in order to analyze OXPHOS and glycolytic parameters. For MCF-7 cells, it was sequentially injected to final concentration 5.5 mM glucose, 1.2 μM Oligomycin A, 0.5 μM FCCP, and finally 2 μM Rotenone with 2 μM Antimycin A. For MDA-MB-231 cells, it was used 5.5 mM glucose, 1 μM Oligomycin A, 1 μM FCCP, 1 μM Rotenone, and 1 μM Antimycin A. Right after the assay is ended, OCR and ECAR from each sample were normalized to the corresponding total protein concentration before calculation of metabolic parameters. Briefly, cells were lysed with SDS Lysis Buffer containing 20 mM HEPES, 2 mM EDTA, 0.5% Triton X-100, 0.1% SDS and 1 mM PMSF at 4°C. Proteins concentration was quantified by Bradford method. For OXPHOS parameters, we used normalized OCR and set the Non-Mitochondrial Oxygen Consumption (Non-MOC) as the minimum rate after Rotenone/antimycin A injection. Basal respiration was the last rate after glucose injection minus the Non-MOC; Maximal respiration was the maximum rate after FCCP injection minus the non-MOC; Proton Leak was the minimum rate after oligomycin injection minus the Non-MOC. ATP Production Coupled Respiration was the last rate before oligomycin injection minus the minimum rate after oligomycin injection, and Spare Respiratory Capacity was the maximal respiration minus the basal respiration. To analyze glycolysis, we used normalized ECAR. Glycolysis parameter was the maximum rate before oligomycin injection minus the last rate before glucose injection. Glycolytic capacity was the maximum rate after oligomycin injection minus the last rate before glucose injection, and glycolytic reserve was the glycolytic capacity minus the glycolysis parameter. All assays were done in triplicate and repeated three times.