Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism

Abstract

Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs ( Koppenol et al., 2011 ; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements in living cells, utilizing the Seahorse XF24 Extracellular Flux Analyzer (Figure 1). The Seahorse XF24 Extracellular Flux Analyzer continuously measures oxygen concentration and proton flux in the cell supernatant over time ( Wu et al., 2007 ). These measurements are converted in OCR and ECAR values and enable a direct quantification of mitochondrial respiration and glycolysis. With this protocol, we sought to assess basal mitochondrial function and mitochondrial stress of three different cancer cell lines in response to the cytotoxic test lead compound mensacarcin in order to investigate its mechanism of action. Cells were plated in XF24 cell culture plates and maintained for 24 h. Prior to analysis, the culture media was replaced with unbuffered DMEM pH 7.4 and cells were then allowed to equilibrate in a non-CO₂ incubator immediately before metabolic flux analysis using the Seahorse XF to allow for precise measurements of Milli-pH unit changes. OCR and ECAR were measured under basal conditions and after injection of compounds through drug injection ports. With the described protocol we assess the basic energy metabolism profiles of the three cell lines as well as key parameters of mitochondrial function in response to our test compound and by sequential addition of mitochondria perturbing agents oligomycin, FCCP and rotenone/antimycin A. Figure 1.Overview of seahorse experiment.

Keywords: Bioenergetics; Glycolysis; Mitochondrial metabolism; Mitochondrial respiration; Seahorse XF.

Figure 1.. Overview of seahorse experiment

Figure 2.. Plate layout for cell density evaluation.

Shown here is the exemplary seeding layout for the SK-Mel-5 and SK-Mel-28 cell lines (seeding density for HCT-116 cells was evaluated on a second plate; not shown).

Figure 3.. Optimization of assay conditions: evaluation of OCR and ECAR depending on the seeding density of three different cell lines

Figure 4.. Seahorse XF 24 sensor cartridge.

A. The sensor cartridge sitting on top of a calibration plate with injection ports shown. B. Bottom side of the sensor plate which shows sensors with embedded fluorophores.

Figure 5.. Mitochondrial stress test.

OCR was measured after mensacarcin was injected (black arrow) in different concentrations to SK-Mel-28 cells followed by consecutive injections of oligomycin (1 μM), FCCP (0.5 μM), and antimycin A (0.5 μM)/rotenone (0.5 μM) (n = 3).

Figure 6.. Basal bioenergetic state of SK-Mel-28, SK-Mel-5 and HCT-116 cells.

The basal energy metabolism of each cell line was assessed by analyzing OCR/ECAR ratios. OCR and ECAR were acquired with the same protocol as described above but without the injection of compounds. The protocol commands consisted of one loop with 8 measurements. Several separate assays were performed (n = 25).