Celltaq

We used a Seahorse XFe24 analyzer (Seahorse Bioscience) to real-time analyze the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of NK cells cultured under various conditions. In brief, purified dNK and pNK were cultured for 24 h supplemented with IL-15 as a survival factor, then they were adhered to 24-well plate using Cell-Taq (BD Pharmingen) at 300,000 cells/well. During analysis,10 mM glucose, 0.5 µM oligomycin, and 100 mM 2-deoxyglucose (2-DG) were injected sequentially, being used to determine non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve. Consecutive additions of oligomycin (1 µM), FCCP (1 µM, Sigma), and rotenone (500 nM) plus antimycin A (500 nM) allowed to obtain with accuracy the oxygen consumption due to basal respiration, maximal respiration, ATP production and non-mitochondrial respiration.

An XF-24 Extracellular Flux Analyzer (Seahorse Bioscience) was used for real-time analyses of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of NK cells according to the manufacturer's protocol. Briefly, NK cells were collected after stimulation and resuspended in XF base and assay medium (Agilent Technologies) for ECAR and OCR analysis, respectively. Cells were adhered to CellTaq (BD Pharmingen) coated XF 96-well microplate (Seahorse Bioscience) at 200,000 cells per well. Cells were starved in a non-CO2 chamber at 37°C for 1 h to deplete all the stored glucose in NK cells. ECAR was measured under basal conditions followed by sequential addition of 10 mM glucose, 0.5 μM oligomycin, and 100 mM 2-deoxyglucose (2-DG). This procedure allows an estimation of extracellular acidification caused by non-glycolytic acidification, glycolysis, and glycolytic capacity of NK cells. OCR was measured under basal conditions followed by the injections of oligomycin (1 μM), FCCP (1 μM), and rotenone (500 nM) plus antimycin (500 nM). This protocol allows the accurate calculation of oxygen consumption due to basal respiration, maximal respiration, ATP production and non-mitochondrial respiration.

For real-time analysis of the ECAR and OCR of purified CD8 T cells, a Seahorse XFp Analyzer (Seahorse Bioscience) was used. In brief, 200,000 isolated CD8 T cells were adhered to a CellTaq (BD PharMingen)-coated 8-well XFp Cell Culture Microplate (Seahorse Bioscience). Sequential measurements of ECAR and OCR following addition of the inhibitors (Sigma-Aldrich) oligomycin (2 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (0.5 µM), rotenone (100 nM) plus antimycin A (4 µM), and 2-deoxyglucose (30 mM) were acquired.