Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

1. Preparation of Reagents

1. Prepare 500 mL of PBS by dissolving 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, in ddH₂O. Adjust the pH to 7.4 with HCl. Sterilize by autoclaving.
2. Prepare 100 mL of RPMI medium: RPMI-1640 medium with L-Alanyl-Glutamine supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL).
3. Prepare 50 mL of 0.1 M NaHCO₃ in distilled water. Adjust the pH to 8.1, filter sterilize (0.22 μm) and store at 4 °C.
   NOTE: For two 8-well extracellular flux analyzer plates, prepare 250 μL of 0.1 M NaHCO₃ pH 8.1.
4. Prepare 1 mL of 2 M D-glucose in distilled water. Filter sterilize (0.22 μm) and store at -20 °C.
5. Prepare 100 μL of 1 mM Oligomycin A in ethanol. Filter sterilize (0.22 μm) and store at -20 °C.
6. Prepare 250 μL of 1 M 2-deoxy-D-glucose (2-DG) in Minimal DMEM (Dulbecco's Modified Eagle's medium). Warm to 37 °C and adjust the pH to 7.4 (carry out pH measurement at 37 °C). Filter sterilize (0.22 μm) and store at -20 °C.
7. Prepare 100 μL of 1 mM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in DMSO. Filter sterilize (0.22 μm) and store at -20 °C.
8. Prepare 100 μL of 1 mM Rotenone in ethanol. Filter sterilize (0.22 μm) and store at -20 °C.
9. Prepare 100 μL of 1 mg/mL Antimycin A in ethanol. Filter sterilize (0.22 μm) and store at -20 °C.
10. Just before the use, prepare 10 mL of Glycolysis stress test medium. Warm Minimal DMEM to 37 °C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 °C).
11. Prior to the use, prepare 10 mL of Cell Mito stress test medium with BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose, 1 mM HEPES (pH 7.4), 1 mM pyruvate and 0.1 % BSA (bovine serum albumin). Warm to 37 °C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 °C).
12. Prior to the use, prepare 10 mL of Cell Mito stress test medium without BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose,1 mM HEPES (pH 7.4) and 1 mM pyruvate. Warm Cell Mito stress test medium without BSA to 37 °C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 °C).
    NOTE: BSA is added to the Cell Mito stress test medium because the cells respond better to FCCP when the medium is supplemented with BSA (different conditions were tested). Cell Mito stress test medium without BSA is used for loading the ports as the manufacturer does not recommend using BSA in the medium.

2. Overnight Cultivation of Mononuclear Cells

NOTE: Perform all sub-steps in a sterile tissue culture hood.

1. Prepare two T75 flasks with 20 mL of RPMI. To each flask, add 30 x 10⁶ isolated mononuclear cells. Incubate the cells with the flask standing up for 16–24 h at 5% CO₂ and 37 °C.

3.  Preparation of Cell Adhesive-coated Plates

1. Coat two 8-well extracellular flux analyzer plates.
   NOTE: Perform the coating in a sterile tissue culture hood.
2. Add 2.2 μL of cell adhesive (density: 2.54 mg/ml) to 250 μL of 0.1 M NaHCO₃, pH 8.1, and pipette immediately 12.5 μL of the solution into each well.
   NOTE: Cell adhesive stock solutions can differ in their density, adjust the volume added to NaHCO₃ accordingly.
3. Let the plates sit in the hood for about 20 min, then aspirate the cell adhesive and wash each well twice using 200 μL of sterile water. Let it sit in the hood with the lid open until wells are dry.
4. Use the plates right away or save up to 1 week at 4 °C with the rim wrapped in paraffin film to avoid condensation. Ensure that the plates are warmed up to room temperature (for about 20 min) in the hood before seeding the cells.

4. Hydration of Sensor Cartridge

NOTE: Hydrate two 8-well extracellular flux analyzer cartridges.

1. Separate the utility plate and the sensor cartridge. Place the sensor cartridge upside down on the lab bench.
2. Fill each well of the utility plate with 200 μL calibrant. Fill each moat around the outside of the wells with 400 μL of calibrant.
3. Return the sensor cartridge to the utility plate that now contains the calibrant.
4. Place the cartridge assembly in a humidified, non-CO₂, 37 °C incubator overnight.
5. Turn on the extracellular flux analyzer and let it warm to 37 °C overnight.

5. Seeding Cells in Cell Adhesive-coated Plates

NOTE: For Glycolysis stress test, use Glycolysis stress test medium. For Cell Mito stress test, use Cell Mito stress test medium with BSA.

1. Seed cells for the Glycolysis stress test and the Cell Mito stress test in separate plates. For each test, use cells from one flask with overnight culture.
2. Centrifuge cells at 200 x g for 5 min at room temperature. Resuspend cells in 1 mL of the appropriate medium and count them.
3. Add 4 x 10⁶ of live cells to the final volume of 400 μL (use appropriate medium).
4. Plate 50 μL of the cell suspension into wells B-G. Ensure 500,000 cells are seeded in one well.
   NOTE: It is crucial to seed exactly 500,000 cells per well as no other normalization is performed. That way, the results from different patients can be compared. The optimal number of replicates is six, as is described here. Using less replicates is not recommended since primary cells could sometimes behave erroneously.
5. Add 180 μL of the appropriate medium into wells A and H (these wells will serve as a background correction).
6. Centrifuge the plate at 400 x g for 5 min at room temperature with brake set to 1.
7. Add 130 μL of appropriate medium to wells B-G in two 8-well extracellular flux analyzer plates slowly and carefully. Visually confirm that the cells are stably adhered to the bottom of the wells by viewing under the microscope.
8. Place the plate into a humidified, non-CO₂, 37 °C incubator for 30 min.

6. Loading the Sensor Cartridge

1. For the Glycolysis stress test, prepare 250 μL each of 100 mM glucose, 20 μM Oligomycin A and 1 M 2-DG, all in Glycolysis stress test medium.
2. For the Cell Mito stress test, prepare 250 μL each of 20 μM Oligomycin A, 15 μM FCCP, 30 μM FCCP and a mixture of 10 μM Rotenone and 10 μg/ml Antimycin A, all in Cell Mito stress test medium without BSA.
   NOTE: Injecting two concentrations of FCCP in one assay is recommended since there is not enough patients' material for the FCCP titration. Nevertheless, the concentrations should be determined by the researcher.
3. Load the compounds into the appropriate injector ports of the cartridge as follows (Table 1):

7. Setting Up the Program

1. For the Glycolysis stress test, set up the program as described in Table 2.
2. For the Cell Mito stress test, set up the program as described in Table 3.
3. Start the program. Replace the calibrant plate with the assay plate (when prompted).

Table 1: Compound volumes.

Table 2: Program for Glycolysis stress test.

Table 3: Program for Cell Mito stress test.