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 |
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).