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

1. 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.
2. 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.
3. 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.
4. Prepare a 400 µM solution of AMP, ADO and INO by mixing 1 ml of the 2 mM stock solution in 4 ml of serum-free medium (5 ml final volume).
   1. Make 1:4 dilution of each 400 ....

Results

Figure 1. Generation of an internal standard curve. Representative calibration standard curve for ADO and the relative equation obtained. Please .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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