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

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Citazione di questo articolo
Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells. J. Vis. Exp. (Pending Publication), e20267, doi: (2023).

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