Source: Hlozkova K. et al. Assessment of the Metabolic Profile of Primary Leukemia Cells. J. Vis. Exp. (2018).
This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.
1. Preparation of Reagents
2. Overnight Cultivation of Mononuclear Cells
NOTE: Perform all sub-steps in a sterile tissue culture hood.
3. Preparation of Cell Adhesive-coated Plates
4. Hydration of Sensor Cartridge
NOTE: Hydrate two 8-well extracellular flux analyzer cartridges.
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.
6. Loading the Sensor Cartridge
7. Setting Up the Program
Port | Glycolysis stress test | Cell mito stress test | ||
Load to the port | Final concentration in the wells | Load to the port | Final concentration in the wells | |
A | 20 μL of 100 mM glucose | 10 mM glucose | 20 μL of 20 μM Oligomycin A | 2 μM Oligomycin A |
B | 22 μL of 20 μM Oligomycin A | 2 μM Oligomycin A | 22 μL of 15 μM FCCP | 1.5 μM FCCP |
C | 25 μL of 1 M 2-DG | 100 mM 2-DG | 25 μL of 30 μM FCCP | 4.5 μM FCCP |
D | X | 25 μL of 10 μM Rotenone and 10 μg/ml Antimycin A | 1 μM Rotenone and 1 μg/ml Antimycin A |
Table 1: Compound volumes.
Step | Settings |
Calibration | Automatic |
Equilibration | Automatic |
Baseline measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port A | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port B | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port C | Injection |
Measurement | Four times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Table 2: Program for Glycolysis stress test.
Step | Settings |
Calibration | Automatic |
Equilibration | Automatic |
Baseline measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port A | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port B | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port C | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Injection of the port D | Injection |
Measurement | Three times: Mix – 3 min, Wait – 0 min, Measure – 3 min |
Table 3: Program for Cell Mito stress test.
The authors have nothing to disclose.
Seahorse Analyzer XFp | Agilent Technologies | S7802A | |
Seahorse XFp Cell Culture Miniplate | Agilent Technologies | 103025-100 | |
Corning™ Cell-Tak Cell and Tissue Adhesive | ThermoFisher Scientific | CB40240 | |
D-(+) Glucose | Sigma-Aldrich | G7021-100G | |
Oligomycin A | Sigma-Aldrich | 75351-5MG | |
2-Deoxy-D-glucose | Sigma-Aldrich | D8375-1G | |
FCCP | Sigma-Aldrich | C2920-10MG | |
Rotenone | Sigma-Aldrich | R8875-1G | |
Antimycin A from Streptomyces sp. | Sigma-Aldrich | A8674-25MG | |
Seahorse XF Base Medium, 100mL | Agilent Technologies | 103193-100 | |
L-glutamine solution, 200 mM | Sigma-Aldrich | G7513-100ML |