Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

The day before the experiment, turn on the analyzer, and let it warm up to 37 degrees Celsius. Open the sensor cartridge package, and separate the cartridge from the utility plate. Then, add 200 microliters of the calibrant solution to each well of the utility plate, and put the sensor cartridge back in, making sure that the sensors are completely submerged.

For optimum results, incubate the cartridge overnight at 37 degrees Celsius in a carbon dioxide-free incubator that is properly humidified. To prepare an adhesive-coated plate, pipette 25 microliters of the cell adhesive solution to each well of the plate, and incubate it at room temperature for 20 minutes. Then, remove the solution, and wash the plate twice with 200 microliters of sterile water per well.

Keep the plate open for 15 minutes inside a cell culture hood to allow the wells to dry. Centrifuge the previously isolated NK cells at 200 times g for five minutes. Then, remove supernatants and wash the cells in warmed mitochondrial stress test medium, or glycolysis stress test medium. Pellet the cells again, and resuspend them to the preferred cell concentration in the same medium. Plate 180 microliters of cell suspension per well into the assay plate.

Use wells A1, A12, H1, and H12 as control wells for background correction, adding 180 microliters of the assay medium to these wells. Incubate the plate for 30 minutes at 37 degrees Celsius in a carbon dioxide-free incubator. Centrifuge the plate at 200 times g for five minutes. Then, observe the cells under the microscope to check that they form a monolayer at the bottom of the well.

Incubate the cells for another 25 minutes. Warm working solutions to 37 degrees Celsius and readjust their pH to 7.4, if needed. Load the previously-prepared compounds for a mitochondrial stress test, or for a glycolysis stress test, into ports A, B, and C of the hydrated sensor cartridge. Put the loaded sensor cartridge back in the incubator while setting up the program. To set up the extracellular flux assay protocol, open the software, and use group definitions to define the pre-treatment conditions.

Use the Plate Map tab to indicate the groups of wells that have similar conditions. Also, indicate the background correction and empty wells. Then, set the program in the Protocols tab. Start the program and place the sensor cartridge and utility plate onto the tray. After the calibration step, replace the calibrant plate for the assay plate with the attached cells. Once the run is completed, retrieve the data and analyze it.