Xf96 cell culture 96 well plate

We seeded 10 × 10³ MDA-MB-231 cells per well in 80 μL of complete medium in a Seahorse Bioscience (North Billerica, MA) XF96 cell culture 96-well plate. The next day we replaced the medium with 1 of 4 of the following different media and drug conditions:

Glutamine media: phenol red-free DMEM, 1% FBS, 0.1 nM estrogen, 1% penicillin/streptomycin, 1% pyruvate, and 2.5% glutamine

Glutamine media plus 40 mM DCA

No glutamine media: phenol red-free DMEM, 1% FBS, 0.1 nM estrogen, 1% penicillin/streptomycin, and 1% pyruvate

No glutamine media plus 40 mM DCA

The following day we performed a glycolysis stress test using the Seahorse Bioscience XF Analyzer. Before running the experiment we replaced the media with the Seahorse XF glycolysis stress test assay medium, excluding glutamine in conditions 3 and 4. After incubating in a CO₂-free incubator for an hour, we added 10 mM glucose, 2 mM oligomycin, and 100 mM 2-deoxyglucose to the injection ports. After calibrating the analyzer, we ran the experiment for a total time of 80 minutes.