This protocol focuses on quantifying mitochondrial respiration of mouse and human neutrophils, as well as neutrophil-like HL60 cells using the metabolic extracellular flux analyzer. This method allows us to quantify mitochondrial function in neutrophils under normal and disease conditions. This is a straightforward technique that can deliver insights into the mitochondrial function of the cells in response to drugs or any disease condition.
Since different cells have different mitochondrial respiratory efficiency, optimization of cell seeding density and reagent concentration has to be performed accurately to get high-quality results. To begin the metabolic extracellular flux assay, take a sensor cartridge packed over a specially designed 96-well plate and transfer 200 microliters of calibration medium into each well of the underlying plate. Carefully lift the cartridge and the plate with calibrant, and place it in a non-carbon-dioxide humidified 37 degrees Celsius incubator overnight for hydration.
Next, based on the cell type, coat the 96-well plate with a specific coating to ensure cell adhesion. Afterward, wash the plates with 200 microliters of sterile PBS twice. Prepare the assay medium in DMEM with one millimolar pyruvate, 10 millimolar glucose, and two millimolar glutamine.
Procure the mouse and human neutrophil cells as described in the text manuscript. For the neutrophils procured from the mouse, adjust the cell density to 1.1 times 10 to the sixth cells per milliliter by adding assay medium. Then seed 180 microliters of the mouse neutrophil suspension per well into the prepared 96-well plate.
Similarly, adjust the human neutrophil cell density to 2.2 times 10 to the sixth cells per milliliter using the assay medium. On the day of the assay, count the neutrophil-like HL60 cells manually using the hemocytometer. Then centrifuge the differentiated or undifferentiated HL60 cells at 300G at room temperature for five minutes.
Wash the cells with DMEM once and re-suspend them in the assay medium to achieve a cell density of 1.39 times 10 to the sixth cells per milliliter. Next seed 180 microliters of differentiated or undifferentiated HL60 cells into the pre-prepared 96-well plate containing the mouse and human neutrophil samples. Finally, add complete medium to the corner wells of the cell culture plate devoid of cells for background correction while seeding.
Centrifuge the plate at 300G at room temperature for three minutes without break to ensure proper adherence of the cells on the bottom of the plate. Finally, incubate the plate for one hour to pre-equilibrate the cells with the assay medium. Open the mitochondrial stress test kit and prepare the reagents as per the manufacturer's instructions.
Next, load the cartridge with reagents, starting with the prepared oligomycin into port A, FCCP into port B, and rotenone/antimycin A mixture into Port C for injections. Fill the unused ports with 20 microliters of assay medium. Equilibrate the assay system at 37 degrees Celsius at least five hours before the assay by opening the Wave software and clicking the Heater On tab.
After reaching the temperature, the lower left corner of the Wave software shows ready. Open a template for the mitochondrial stress assay kit in the software. Select injection strategies on the left side, then click Add and name the injection condition as human neutrophils or mouse neutrophils or HL60.
Select port A to D by clicking on each, and click on Add Compound and enter oligomycin or FCCP or rotenone/antimycin A with the respective concentrations. Select Pretreatment on the left side, then click Add and name them CD18 and Cell Tak separately. Select the Cell Type on the left side.
Then click Add and name them human neutrophils, mouse neutrophils, and HL60 or DHL60 with a feeding density as applicable. Define the groups by clicking on Add Group. Then click on the underlined definition.
Click on Plate Map in the top menu to observe all the groups with definitions on the left side and the plate map on the right. Drag and drop to add each well to the group while maintaining the four corner wells as background. To set up the protocol, select the Protocol tab in the top menu, and define three cycles of baseline, oligomycin, FCCP, rotenone and antimycin A mixture, by setting the time to mix as three minutes, rest as zero minutes, and measurement as seven minutes.
Go to the Run Assay page, provide the project summary information for reference, and click Start Run. Give the location to save the files so that all the results are saved after completion of the assay. Then click on Start Run again.
After the automatic loading of the assay, wait for the tray to open to place the sensor cartridge and plate with 200 microliters of calibrant. Set the cartridge barcode direction facing to the right. Run the calibration by clicking I'm Ready, which takes approximately 20 minutes.
After the calibration, click on Open Tray, replace the plate with the cell seeded plate, and click on I'm Ready to continue the assay. After completing the assay, the data is automatically saved. Export the results to a spreadsheet or other analysis software file.
Plot the graph and analyze the data. The representative dynamics of oxygen consumption rate or OCR indicated changes in the mitochondrial respiration in mouse and human neutrophils, as well as undifferentiated and differentiated HL60 cells in response to oligomycin, FCCP, and the mixture of rotenone and antimycin A.The OCR values decreased in all cells after oligomycin treatment due to the inhibition of the proton channel of ATP synthase. FCCP treatment increased the flow of electrons and oxygen consumption to restore the OCR value and achieve maximal respiration.
A mixture of rotenone and antimycin A blocked complexes 1 and 3 of the electron transport chain, leading to the elimination of mitochondrial respiration. Decreased mitochondrial respiration was observed in HL60 cells after neutrophil-directed differentiation. Differentiated HL60 cells were found to have significantly lower basal mitochondrial respiration, proton-leak-linked respiration, ATP-linked respiration, and non-mitochondrial respiration.
The increase in maximal respiration of differentiated HL60 cells was not substantial, while the spare respiratory capacity was significantly increased. Make sure the cells are at the bottom of the plate, as the sensor cartridge should not have any obstacles due to the floating cells while reading the OCR value. This technique would help researchers to study the effect of drugs or mitochondrial dysfunction in neutrophil-associated studies.
