Xf96 plate

20,000 Calu-3 cells per well were seeded into a Seahorse XF96 plate and incubated at 37 °C, 5% CO2 for 48 hours. The medium was changed 24 hours prior to Seahorse experiment with or without 100 μg/mL LPS. Three days post-seeding, the Seahorse Glycolysis Stress Assay was performed according to the manufacturer’s instructions with the injection of 5 or 15 mM Glucose. The plate layout was separated into quadrants to reduce edge effects.
For LPS experiments, viable cells were quantified after the seahorse assay using CyQuant Direct according to the manufacturer’s instructions. Briefly, the cells’ supernatant was partly aspirated to reach a volume of 100 μL, then 100 μL of the freshly prepared staining solution was added to the cells. After incubation for 1 h at 37 °C without CO2, fluorescence was read with bottom reading mode of a standard plate reader.

To compare the effects of TNFα, IL-1β, IL-6 and vehicle on primary hypothalamic neuronal extracellular flux, cultured primary hypothalamic neurons seeded on XF96-PS plates were treated with TNFα (5 nM, R&D systems, 410-MT, UK), IL-1β (5 nM, R&D systems, 410-ML), IL-6 (5 nM, R&D systems, 406-ML, UK) or 0.5% BSA (vehicle control, Thermo Fisher) for 16 h at 37 °C. Cells were then washed with XF assay medium containing 25 mM glucose (pH adjusted to 7.5) and incubated for 1 h in a 37 °C air incubator. The XF96 plate (Seahorse Bioscience) was then transferred to a temperature-controlled (37 °C) Seahorse (extracellular flux) analyser (Seahorse Bioscience) and subjected to an equilibration period. One assay cycle comprised a 1 min mix, 2 min wait and 3 min measure period. After four basal assay cycles, oligomycin (1 μg ml⁻¹) was added by automatic pneumatic injection to inhibit the ATP synthase and thus approximate the proportion of respiration used to drive ATP synthesis and proton leak. After three further assay cycles, carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (0.5 μM) was added the same way to stimulate maximal respiration in mitochondria by chemical uncoupling. After another three assay cycles, rotenone (4 μM) plus antimycin A (2 μM) was added to inhibit the respiratory chain and determine the non-mitochondrial respiratory rate.

Cells were plated in a Seahorse XF96 plate at a density of 15,000 cells per well, and the compounds that included glucose, oligomycin, and 2-deoxy-d-glucose (2-DG) were loaded into appropriate ports of a hydrated sensor cartridge. Finally, the cells’ glycolysis stress was tested using the Seahorse XFe/XF Analyzer.