Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells
Published: April 30, 2023
Abstract
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.
Protocol
1. Preparation of Reagents 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. 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). Prepare 50 mL of 0.1 M NaHCO3 in distilled water. Adj…
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
Citazione di questo articolo
Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells. J. Vis. Exp. (Pending Publication), e20267, doi: (2023).