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

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.

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