2.7. Determination of Energetic Metabolism

The oxygen consumption rate (OCR) in HT-29 cells was measured in real-time using a XF-24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) as previously reported [17]. For this, 3 × 10⁴ cells were seeded for 16 h in the XF-24 plate and treated for 48 h on 0.5 mmol L⁻¹ of FeNPs or VENOFER^®. The medium was replaced with 450 μL/well of XF-24 running media (Seahorse Bioscience, Billerica, MA, USA), supplemented with 25 mmol L⁻¹ glucose, 2 mmol L⁻¹ glutamine, 1 mmol L⁻¹ sodium pyruvate, without serum and pre-incubated at 37 °C for 20 min in the XF Prep Station incubator (Seahorse Bioscience, Billerica, MA, USA) in the absence of CO₂. The plate was then transferred to the XF-24 Extracellular Flux Analyzer, and after an OCR baseline measurement, four sequential injections of compounds that affect bioenergetics were performed, as follows: 55 μL of oligomycin (1 μg mL⁻¹), 61 μL of 2,4-Dinitrophenol (2,4 DNP) (1 mmol L⁻¹), and 68 μL of antimycin A/rotenone (10 μmol L⁻¹/1 μmol L⁻¹) at injection in port C. Each treatment was carried out in three replicates and the final results were expressed as pmol of O₂ consumed per 105 cells per minute (pmol O₂/105 cells/min).

To evaluate glycolysis, extracellular acidification rate (ECAR) was measured in real-time using a XF-24 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA) as previously reported [17]. ECAR was measured after addition of 55 μL of rotenone (1 μmol L⁻¹), 61 μL of glucose (30 mmol L⁻¹) and 68 μL 2-Deoxy-d-glucose (2-DG) (100 mmol L⁻¹). Prior to measure ECAR, HT-29 cells were treated as in previous assay per triplicate and average ECAR value, expressed as milli-pH per minute (mpH/min) per 3.0 × 10⁴ cells, was calculated for each treatment.