Extracellular Purine Metabolism: An HPLC-based Assay to Measure Adenosine Generation and Metabolism in Leukemic Cells

Published: April 30, 2023

Abstract

Source: Serra S et al. HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells. J. Vis. Exp. (2016)

This video explains a quantitative reverse phase high-performance liquid chromatography (RP-HPLC) method to measure metabolism of adenosine and associated purines generated by consumption of AMP in extracellular environment of leukemic cancer cells. The protocol describes a sensitive, specific, reliable and reproducible RP-HPLC assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions.

Protocol

1. Preparation of Standard and Inhibitors Stock Solutions To prepare AMP, weigh out 0.00345 g of AMP and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of AMP. To prepare ADO, weigh out 0.00265 g of ADO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of ADO. To prepare INO, weigh out 0.00268 g of INO and dissolve it in 5 ml of serum-free medium to have a 2 mM standard solution of INO. Prepare a 400 µM solution of AMP, …

Representative Results

Figure 1. Generation of an internal standard curve. Representative calibration standard curve for ADO and the relative equation obtained.  Please click here to view a larger version of this figure.

Divulgaciones

The authors have nothing to disclose.

Materials

AIM V serum free medium  GIBCO  12055-091  Liquid (research grade)
adenosine 5’-monosphate (AMP)  Sigma-Aldrich  A1752
adenosine (ADO) Sigma-Aldrich  A9251
inosine (INO)  Sigma-Aldrich  I4125
acetonitrile (CHROMASOLV Plus)  Sigma-Aldrich  34998 HPLC-grade
ammonium acetate  Sigma-Aldrich  9688 7 mM, pH 3.0
short thread vials  VWR  548-0029  1.5 ml/glass
micro-inserts  VWR  548-0006  0.1 ml/glass
screw caps  VWR  548-0085  9 mm/PP blue
Atlantis dC18 Column  Waters  186001344 5 µm, 4.6 mm x 150 mm
Atlantis dC18 Guard Column  Waters  186001323 5 µm, 4.6 mm x 20 mm
Waters Alliance 2965 Separations Module  Waters  HPLC separation module
Waters 2998 Photodiode Array (PDA) Detector  Waters  UV detector
Waters Empower2 software  Waters

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Citar este artículo
Extracellular Purine Metabolism: An HPLC-based Assay to Measure Adenosine Generation and Metabolism in Leukemic Cells. J. Vis. Exp. (Pending Publication), e20279, doi: (2023).

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