Seahorse xfp cell energy phenotype test kit

Simultaneous measurement of cell oxygen consumption rate (OCR) and intracellular acidification rate (ECAR) was performed in real time using a Seahorse Metabolic Analyzer XFp (Agilent Technologies, Santa Clara, CA). hTCEpi cells were seeded into Seahorse XFp miniplates and cultured in growth and basal conditions with or without insulin stimulation at 37 °C, 5% CO₂ for 24 hours. Before initiating measurements, cells were incubated for 1 hour at 37 °C with Seahorse XF base medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (pH 7.4) in a non-CO₂ incubator. OCR and ECAR were analyzed using a Seahorse XFp Cell Energy Phenotype Test Kit (Agilent Technologies, Santa Clara, CA) according to manufacturer instructions. Ten μM oligomycin was added to inhibit ATP synthase at 18 minutes followed by three separate injections of 10 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) at 20 minute intervals to alter the mitochondrial membrane potential and allow for maximal oxygen consumption due to uninhibited electron flow through the electron transport chain. Measurements were obtained every 6–7 minutes for a total of 94 minutes. Data were analyzed using the manufacturer provided Wave software, version 2.3.0. The ratio for OCR/ECAR was calculated for each experiment. All assays were performed in triplicate and repeated a minimum of two additional times.

To assess CPB cell energy phenotype alteration post-ISKNV infection, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of ISKNV-infected cells and control cells were determined using Seahorse XFp Cell Energy Phenotype Test Kit according to manufacturer’s protocol, together with Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). At 60 hpi, control and ISKNV-infected CPB cells at equal densities (4 × 10⁴ cells per well) were seeded in XFp Cell Culture Miniplate with 80 μL complete DMEM medium containing 2% FBS at 28 °C with 5% CO₂ overnight. Then, the cell medium was replaced with seahorse XF base medium containing 10 mM Glucose, 1.0 mM Sodium pyruvate and 2 mM Glutamine, and the miniplate was placed in a CO₂-free incubator at 37 °C for 1 h. After incubation, drugs were injected to the final concentration as 50 μM of oligomycin and 50 μM of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and the miniplate was transferred to the seahorse XFp extracellular flux analyzer (Agilent, Santa Clara, CA, USA). The OCR and ECAR were measured and normalized by cell number.

Cell mitochondrial function was evaluated by using the Seahorse XFp Cell Energy Phenotype Test Kit on the Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, CA, USA). Cells were seeded at 20,000 cells per well into XFp well cell culture plates and incubated overnight at 37 °C in a 5% CO₂-humidified atmosphere in Seahorse XF Base Medium (Agilent Technologies) with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cartridge compounds were loaded to obtain a final concentration of 1 μM Oligomycin and 1 μM FCCP. Data were analyzed and visualized using Wave 2.3.0 software (Agilent Technologies), and values of OCR and ECAR were normalized to the total protein levels (Bradford Reagent assay, Sigma-Aldrich) in each well. The experiment was performed with three replicates.