Published: July 20th, 2016
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.
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.
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....
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
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.
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.......
|double deionised water
|purified anti-CD3, -CD14, -CD16
|Dynabeads sheep anti-mouse IgG
|Phosphate-buffered saline (PBS)
|bovine serum albumin (BSA)
|PBS 0.1 % BSA 2 mM EDTA, pH 7.4
|AIM V serum free medium
|liquid (research grade)
|adenosine 5’-diphosphate (ADP)
|adenosine 5’-monosphate (AMP)
|adenosine deaminase inhibitor
|adenosine deaminase inhibitor
|Dimethyl sulfoxide (DMSO)
|nucleoside transporter inhibitor
|acetonitrile (CHROMASOLV Plus)
|7 mM, pH 3.0
|min. 37 %
|Bürker cell counter
|Dynal magnetic bead separator
|microcentrifuge safe-lock tubes
|PET centrifuge tubes
|15 – 50 ml
|Minisart RC4 syringe filters
|Sartorius Stedim Biotech
|membrane 0.2 µm
|short thread vials
|9 mm/PP blue
|Atlantis dC18 Column
|5 µm, 4.6 x 150 mm
|Atlantis dC18 Guard Column
|5 µm, 4.6 x 20 mm
|Waters Alliance 2965 Separations Module
|HPLC separation module
|Waters 2998 Photodiode Array (PDA) Detector
|Waters Empower2 software
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