Seahorse xf assay medium

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured by an XFp extracellular analyzer (Agilent Technologies, Santa Clara, CA, USA). MCF-7 transfected with FoxO3a shRNA were seeded at 3.0 × 10⁴ cells/well density in 8-well plates for overnight incubation to allow adherence to the plate. Then the cells were treated with erastin (16 μM) in glucose depletion medium for 6 h. After 6 h of erastin administration, the cells replaced to Seahorse XF assay Medium (Agilent, Santa Clara, CA, USA) pH 7.4 supplemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. After monitoring 18 min of basal respiration, erastin (16 μM) was added to the system, then sequential injection 1 μM of mitochondrial inhibitors oligomycin, FCCP, and antimycin (AA) plus rotenone (AR) provided by the manufacturer (#101706–100, Agilent Technologies) every 20 min after monitoring. OCR and ECAR was automatically calculated using the Seahorse XFp software. Every point represents an average of three different wells.

B12 cells were plated at a density of 40,000 cells per well in DMEM (5% FBS and 1% pen/strep) in a Seahorse XFe24 cell culture microplate and incubated overnight in the absence or presence of 100 nM DOPPA. All wells were then washed twice with bicarbonate-free Seahorse XF assay medium (Agilent) supplemented with 10 mM glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate, and pH adjusted to 7.35 +/− 0.05. Following washes, the cell culture plate was incubated in the XF assay medium for 1 hour at 37 ^oC. Mitochondrial oxygen consumption rate (OCR) was first measured at baseline, and then sequentially after the administration of 1 µM oligomycin (Agilent), 1 µM FCCP (Agilent), and 0.5 µM rotenone/antimycin A (Agilent). After the Seahorse XF MitoStress Test assay, cells in control and treatment wells were lysed and protein harvested using RIPA buffer as stated earlier. Data was normalized by protein concentration.

Cells were seeded in 6-well plates (200,000 cells/well) and transfected appropriately, as described in figure legends. The day before the assay, cells were detached by trypsinisation, counted, and seeded at 10,000 cells/well in 96-well Seahorse XF cell culture plates (Agilent). The sensor cartridges were hydrated overnight with tissue culture grade H₂O in a non-CO2 incubator at 37 °C as per the manufacturer’s instructions. On the day of the assay, H₂O in the sensor plate was replaced with Seahorse XF Calibrant (Agilent) and cells were washed with HBSS and incubated in Seahorse media (Seahorse XF assay medium (Agilent), 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent), 10 mM D-(+)-galactose). Both sensor and cell plates were incubated in a non-CO₂ incubator at 37 °C for 1 h before the assay. The sensor plate was loaded with oligomycin (15 µM for 1.5 µM final concentration in wells; injector A), CCCP (5 µM for 0.5 µM final; injector B), and antimycin A and rotenone (5 µM/5 µM for 0.5 µM/0.5 µM final; injector C). The sensor plate was calibrated before loading cells and running a mitochondrial stress test using a Seahorse XFe96 (Agilent). Following assays, cells were washed and fixed in 1% (v/v) acetic acid in methanol at −20 °C overnight for SRB assays, which were used for normalising data to protein content.

Control (isotype stimulated), exhaustion (isotype + MICA/MICB + anti-NKp46 stimulated), and NKG2A-tempered (anti-NKG2A + MICA/MICB + anti-NKp46 stimulated) cells were resuspended in Seahorse XF Assay Medium (Agilent Technologies). A total of 1 × 10⁶ cells/well were immobilized with poly-l-lysine (MilliporeSigma). The ECAR and the OCR were measured (pmoles/min) in real time in an Xfe24 analyzer after injection of glucose (10 mM), oligomycin (1 μM), FCCP (1 μM) plus sodium pyruvate (1 mM), and rotenone plus antimycin A (0.5 μM). SRC was calculated from the change from basal oxygen consumption, after addition of glucose, to maximal oxygen consumption, after addition of FCCP. Glycolytic capacity was calculated from the difference between the maximum ECAR following oligomycin injection and the basal ECAR. Glycolytic reserve was calculated from the difference between the maximum ECAR following oligomycin injection and ECAR following glucose injection.

Cells were seeded onto an XFp 8-well Seahorse XFp flux plate (cat#103723-100, Agilent Technologies, Santa Clara, CA, USA) pre-coated with 0.01% poly-L-lysine (cat#P4707, Sigma Aldrich, St. Louis, MO, USA). A cell density of 4000 cells/150 µL per well was seeded and centrifuged at 500× g rpm to encourage adhesion to the plate and form an evenly dispersed monolayer. Cells were then incubated at 37 °C in non-CO₂ conditions in Seahorse XF assay medium (cat#103681-100, Agilent Technologies, Santa Clara, CA, USA) and further processed using the XFp Extracellular Flux Analyzer (cat#431018, Agilent Technologies, Santa Clara, CA, USA) as per the manufacturer’s protocols. The energetic profile of modulated and control cells was determined using Seahorse XFp Extracellular Flux Analyzer and Cell Energy Phenotype Test Kit (cat#103275-100, Agilent Technologies, Santa Clara, CA, USA).

Cells were seeded in six-well plates (200,000 cells/well) and transfected appropriately, as described in figure legends. The day before the assay, cells were detached by trypsinisation, counted, and seeded at 10,000 cells/well in 96-well Seahorse XF cell culture plates (Agilent). The sensor cartridges were hydrated overnight with tissue culture grade H₂O in a non-CO₂ incubator at 37°C as per the manufacturer's instructions. On the day of the assay, H₂O in the sensor plate was replaced with Seahorse XF Calibrant (Agilent) and cells were washed with HBSS and incubated in Seahorse medium [Seahorse XF assay medium (Agilent), 1 mM pyruvate (Agilent), 2 mM glutamine (Agilent) and 10 mM D-(+)-galactose]. Both sensor and cell plates were incubated in a non-CO₂ incubator at 37°C for 1 h before the assay. The sensor plate was loaded with oligomycin (15 µM for 1.5 µM final concentration in wells; injector A), CCCP (5 µM for 0.5 µM final; injector B), and antimycin A and rotenone (5 µM/5 µM for 0.5 µM/0.5 µM final; injector C). The sensor plate was calibrated before loading cells and running a mitochondrial stress test using a Seahorse XFe96 (Agilent). Following assays, cells were washed and fixed in 1% (v/v) acetic acid in methanol at −20°C overnight for sulforhodamine B (SRB) assays, which were used for normalising data to protein content.