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

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

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 pl....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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