HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

Summary

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

Abstract

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (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. The chromatographic separation of adenosine 5'-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

Introduction

Adenosine (ADO) is a purine nucleoside with an adenine molecule attached to a ribose sugar molecule moiety through a glycosidic bond. When present in the extracellular environment, it protects cells from excessive damage by the action of the immune system. This role has been highlighted using different disease models, such as colitis¹, diabetes², asthma³, sepsis⁴, and ischemic injury⁵. One of the main ADO functions is the inhibition of immune responses in the tumor microenvironment, contributing to tumor immune evasion⁶. For this reason, the mechanisms involved in ADO formation and signaling are of cons....

Protocol

CLL blood samples are obtained in accordance with Institutional Guidelines and Declaration of Helsinki.

1. Isolation of Leukemic Lymphocytes from Blood Samples of CLL Patients 

1. Collect blood sample in sodium heparin (green-top) tube¹⁷.
2. Make 1: 3 dilution of whole blood with RT 1x phosphate buffered saline (PBS).
3. Purify peripheral blood mononuclear cells (PBMC) from blood samples by density gradient centrifugation.
   1. Underlay 5 ml of density centrifugation media (e.g., Ficoll) in a 15 ml centrifuge tube and carefully transfer 10 ml of diluted blood. Immediately centrifuge at 1,500 x g for 20 min at RT....

Results

To evaluate the percentage (%) of leukemic cells in freshly purified PBMCs from a representative CLL patient, cells are marked with anti-CD19 and anti-CD5 antibodies. The left panel of Figure 3 represents a cytofluorimetric dot plot with a selective gate on live cells. Figure 3 shows an example of PBMC from a CLL patient before (middle panel) and after (right panel) B cell purification.

An examp.......

Discussion

The protocol described here permits to evaluate the activity of the CD39/CD73 adenosinergic machinery in cell culture media from purified human leukemic cells. Through this HPLC method we can follow and quantitatively measure the enzymatic generation of ADO (CD73-dependent) and its subsequent degradation to INO (CD26/ADA dependent). The use of enzyme inhibitors allows to control the protocol and to have internal controls. The advantages and novelties of this protocol are that i) it may be applied to cells that are growin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human blood
Milli-Q water	Millipore		double deionised water
Ficoll-Paque Plus	GE-Healthcare	17-1440-03
purified anti-CD3, -CD14, -CD16	made in-house		mouse monoclonal
PE-labeled anti-CD19	Miltenyi Biotec	120-014-229
FITC-labeled anti-CD5	Miltenyi Biotec	130-096-574
Dynabeads sheep anti-mouse IgG	Invitrogen	11031
Phosphate-buffered saline (PBS)	Amresco	E404-200TABS	tablets
bovine serum albumin (BSA)	ID bio	1000-70	standard grade
isolation buffer			PBS 0.1% BSA 2 mM EDTA, pH 7.4
AIM V serum free medium	GIBCO	12055-091	liquid (research grade)
adenosine 5’-diphosphate (ADP)	Sigma-Aldrich	A2754
adenosine 5’-monosphate (AMP)	Sigma-Aldrich	A1752
adenosine (ADO)	Sigma-Aldrich	A9251
inosine (INO)	Sigma-Aldrich	I4125
α,β-methylene-ADP (APCP)	Sigma-Aldrich	M8386	CD73 inhibitor
EHNA hydrochloride	Sigma-Aldrich	E114	adenosine deaminase inhibitor
Deoxycoformycin (dCF)	Tocris	2033	adenosine deaminase inhibitor
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2650
Dipyridamole	Sigma-Aldrich	D9766	nucleoside transporter inhibitor
acetonitrile (CHROMASOLV Plus)	Sigma-Aldrich	34998	HPLC-grade
ammonium acetate	Sigma-Aldrich	9688	7 mM, pH 3.0
hydrochloric acid	Sigma-Aldrich	30721-1L	min. 37%
Name	Company	Catalog Number	Comments
Equipment
Bürker cell counter	VWR	631-0920	hemocytometer
DynaMag-15 Magnet	Invitrogen	12301D	Dynal magnetic bead separator
microcentrifuge safe-lock tubes	Eppendorf	030-120-0086	1.5 ml
PET centrifuge tubes	Corning	430053/430304	15 – 50 ml
Minisart RC4 syringe filters	Sartorius Stedim Biotech	17821	membrane 0.2 µm
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