2.12. Seahorse XFp Cell Mito Stress Test

Measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in Caco-2 monolayers was performed by a Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies, California, USA). The day before the assay, the Seahorse XFp Sensor Cartridge was hydrated with XF Calibrant Solution and was kept at 37°C in a CO₂-free incubator overnight. Caco-2 cells were seeded on XFp Miniplates at a density of 1.5 × 10⁴ cells/well. After reaching 100% confluence, cells were treated exactly as described at the FITC-dextran assay. After the treatment, a complete growth medium was replaced with an unbuffered, serum-free Agilent XF Base assay medium, pH 7.4. XFp Mito Stress Test Kit was used to test mitochondrial function. Injection of oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and the mix of rotenone and antimycin A allows determining the key bioenergetic parameters: basal respiration, ATP production-linked respiration (ATP production), maximal respiration, spare respiratory capacity, nonmitochondrial respiration, proton leak, and coupling efficiency. Oligomycin inhibits the F_O subunit of the F_OF₁-ATP synthase, thereby indicating ATP-linked OCR, i.e., level of ATP synthesis. ATP-linked respiration is calculated by the difference between baseline OCR and OCR after oligomycin injection. Distracting nonmitochondrial respiration from the OCR after FCCP injection represents maximal respiration. FCCP is a mitochondrial uncoupler, which separates the activity of phosphorylation and oxidation. Under these circumstances, ETC might work with its maximum rate and consumes higher amounts of O₂ without developing membrane potential between the two sides of the mitochondrial inner membrane. Spare respiratory capacity is defined by the difference between maximal and basal respiration. The mixture of rotenone and antimycin A inhibits CI and CIII, respectively; thus, mitochondrial ETC and O₂ consumption are blocked. The final concentrations of the modulators were 1 μM. OCR after rotenone/antimycin A injection represents nonmitochondrial respiration. ATP-linked respiration divided by basal respiration reveals coupling efficiency.