In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.
This method allows for the determination of natural killer cell metabolism. A key parameter in the activation by measuring oxygen consumption and pH changes in the extracellular medium. The advantage of the extracellular flux analyzer is that it is fully automated and able to test up to 92 samples in real time with low quantities of cells.
Thus allowing high throughput screens. A valid method to determine glycolysis and respiration in inky cells is very important in the clinic. Since there are many diseases including obesity and cancer where inky cell metabolism is impaired.
Begin by pipetting 20 milliliters of lymphocyte separation medium into a 50 milliliter conical tube. While keeping the tube at 30 degree angle, gently pipette 20 milliliters of blood over the medium. Creating a visible and well-defined interface between the two fluids.
Centrifuge the tubes for 25 minutes at 1000 times G.Then carefully take the tubes out of the centrifuge and place them in a rack. Check for the presence of a conspicuous layer of cells at the interface between LSM and plasma. Gently aspirate the mononuclear cell layer with a 10 milliliter plastic pipette and place it in a new 50 milliliter conical tube.
Wash the mononuclear cells twice with 45 milliliters of PBS. And centrifuge them at 800 times G for five minutes. To isolate the natural killer cells or engaves, count the PVM seas and resuspend them in NK separation buffer at one times 10 to the eighth cells per milliliter.
Then transfer 10 milliliters of the cell suspension into a new 50 milliliter tube. Add 500 microliters of NK cell isolation antibody mix. And 10 microliters of anti CD three positive isolation antibody mix to the PBMCs.
And incubate them at room temperature for 10 minutes. Vortex magnetic beads and add one milliliter to the PBMCs and antibodies. Then incubate the MIGS for 10 minutes at room temperature with occasional stirring.
Add 35 milliliters of NK isolation buffer and place the tube on the magnet for 15 minutes. Carefully collect the supernatant with a 50 milliliter plastic pipette without touching the sides or the bottom of the tube. Count the cells and centrifuge them at 800 times G for five minutes.
To stimulate the NK cells with soluble aisle 15, resuspend 750, 000 cells in 100 microliters of IMDM containing 10%HS in a well of a 96 well plate. Dilute human aisle 15 to one microgram per milliliter in the IMDM and 10%HS. And add 100 microliters of the diluted human aisle 15 to the cells.
Repair a control sample with unstimulated cells and place both samples in the incubator at 37 degrees Celsius. Stimulate the cells for 48 hours then perform the extracellular flux assay. The day before the experiment, turn on the analyzer and let it warm up to 37 degrees Celsius.
Open the censor 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 central 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. Which properly humidified. To prepare an adhesive coated blade, pipette 25 microliters of the cell adhesive solution to each well of the plate.
And incubated 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 the cell culture hood to allow the wells to dry.
Centrifuge the previously isolated NK cells add 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.
Lay 180 microliters of cell suspension per well into the assay plate. Use wells A one, A 12 H one and H 12 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 mano layer 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 boards A, B and C of the hydrated censor cartridge Put the loaded censor 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 pretreatment 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.
In order to assess the purity and viability of the NK cells. Small Alec Watts from PVM seas and the isolated NK cell population were stained and analyzed by flow cytometry. The purity of the NK cell population was established by double staying against CD three and CD 56 or NKP 46.
Lots of mitochondrial oxygen consumption rate for the human NK cells are shown here. As expected I cell numbers displayed higher OCR values. Cell numbers also correlated linearly with the amount of DNA or protein in the well.
On the other hand higher cell numbers were not optimal. Because upon addition of DNP, the oxygen concentration in the well was totally depleted in each cycle. Which prevented the accurate calculation of the OCR.
In human NK cells, 100 micromolar DNP was found to be the optimal dose. A typical mitochondrial stress test experiment with 750, 000 NK cells per well is shown here. In this test Oligomycin leads to a dramatic decrease in oxygen consumption.
And an increase in ECAR. Which represents a switch to glycolysis and an effort to maintain cellular ATP levels. Activation of the NK cells by aisle 15 caused an increase in both mitochondrial oxygen consumption and extracellular acidification.
Basal Maximal and ATP linked respiration increased but not proton leak or non mitochondrial respiration. Furthermore, the OCR/ECA rate decreased Indicating a shift to a glycolytic metabolism after IL 15 stimulation. When performing this protocol, it is very important to obtain a pure and healthy cell population to determine the optimal cell number and to titrate the concentration of uncablers.
