Metabolomic studies using Seahorse flux analyzer

Primary bovine chondrocytes were plated at 8.0 × 10⁴ cells/well into specially designed 96-well Seahorse XF cell culture microplates. The confluent monolayers were preincubated for 24 h with or without 2 ng/ml IL1β and with or without 2 mM 2DG in control glucose or galactose culture medium. The medium was changed to serum-free Seahorse XF Base Medium (without phenol red but with 10 mM glucose, 1.0 mM pyruvate, and 2.0 mM glutamine added) or Seahorse XF DMEM, pH 7.4, cells, depending on the assay. Assay medium also contained fresh IL1β. The cells were then mated with a sensor cartridge and analyzed in a Seahorse XFe 96 flux analyzer (Agilent Tech) for real-time detection of changes in proton accumulation and oxygen consumption following the manufacturer’s guidelines. The Seahorse Assays took advantage of five reagents: Oligomycin, FCCP, Rotenone, antimycin A, and 2DG to measure cell metabolism. Oligomycin prevents the increase in mitochondrial respiration induced by ADP without inhibiting uncoupler-stimulated respiration. FCCP is a potent uncoupler of mitochondrial oxidative phosphorylation. It disrupts ATP synthesis by transporting protons across the mitochondrial inner membrane, interfering with the proton gradient. Rotenone and antimycin A are known to inhibit complexes I and III of the electron transfer chain in the mitochondria. 2DG inhibits glycolysis by blocking hexokinase.

Briefly, for the Agilent XF cell energy phenotype test, a combined injection of oligomycin (2 μM final) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) (0.25 μM final) were applied after the instrument completed measurement of basal values. When performing an Agilent XF Cell Mito Stress Test, timed sequential injections of oligomycin (2 μM final) followed by FCCP (0.25 μM final) and, last, a 1:1 mixture of antimycin A (0.50 μM final) with rotenone (0.50 μM final) were applied after measurement of basal values. To perform an Agilent XF Glycolytic Rate Assay, timed sequential injections of a 1:1 mixture of antimycin A with rotenone (0.50 μM final) and, last, 2DG (50 mM final) were applied after measurement of basal values. For the Agilent XF Real-Time ATP Rate Assay, timed sequential injections of oligomycin (1.5 μM final) followed by a 1:1 mixture of antimycin A with rotenone (0.50 μM final) were applied after measurement of basal values. Algorithms provided in Agilent assay report generator Excel files were used to generate blots and bar graphs. Mitochondrial ATP production derived from the Mito Stress test is expressed as OCR (pmol of O₂/min); in the software, ATP production in these OCR units is closely proportional to true ATP values. A measure of ATP production rate in terms of pmol/min ATP is provided by the ATP rate assay. We determined that our culture conditions, namely plating of chondrocytes at a high confluence density in 6-well culture plates, followed by 24 h treatment with or without IL1β, 2DG, or galactose, did not affect any changes in the cell number of primary chondrocytes as measured by Hoechst 33342 dye staining analysis, direct cell counting or BSA assay for protein content of lysates (Figs. 2 and ^S5). As such, no additional normalization of cell counts was taken at the end of Seahorse analyses.