Xf dmem medium ph 7

The extracellular acidification rate (ECAR), which indicates glycolytic activities was analyzed using the Seahorse Glycolysis Stress Test (Agilent) on a Seahorse XFe96 Analyzer (Agilent) according to the manufacturer’s protocol. Briefly, conditioned NK cells were harvested, washed using prewarmed supplemented RPMI medium, and plated on XF96 cell culture microplates (Agilent) coated with poly-d-lysine (Thermo Fisher Scientific) to adhere the cells on the microplate surface. Prior to the assay, the RPMI medium was replaced with XF DMEM Medium, pH 7.4 (Agilent), supplemented with 2 mM glutamine (Agilent). At indicated time points, ECAR was measured in the basal condition and in response to 10 mM glucose, 6 mM oligomycin and 50 mM 2-DG (Agilent).

We used the Seahorse XFe-96 analyzer (Agilent Technologies, Santa Clara, CA, USA) to evaluate the effect of graphene on cellular metabolic function. Cells were treated for eight weeks with different concentrations of graphene, then harvested and seeded in the Seahorse XF cell culture microplates at a density of 15,000 cells per well one day prior to measurement. Before the measurement, we replaced the cell culture medium with the assay medium (bicarbonate-free XF DMEM Medium pH 7.4, Agilent Technologies, Santa Clara, CA, USA) supplemented with 4 mM L-glutamine, 1 mM pyruvate, and 1 g/L D-glucose. The cells were then incubated in a CO₂-free incubator for one hour at 37 °C. After measuring the basal respiration, we performed a mitochondrial stress test by sequential additions of 1 μM oligomycin, 1.2 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 μM rotenone-antimycin A mixture. The differences between OCR (oxygen consumption rate) values in response to respiratory modulators were used to calculate mitochondrial respiratory parameters such as ATP-related respiration and spare respiratory capacities. The changes in ECAR (extracellular acidification rate) were used to calculate glycolytic parameters (spare glycolytic capacity).