Assaying Myeloperoxidase Inhibitors and Hypochlorous Acid Scavengers in HL60 Cell Line Using Quantum Dots

^a Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore

^b Department of Anatomy, National University of Singapore, 4 Medical Drive, Singapore.

^c Department of Epidemiology and Public Health, National University of Singapore, 16 Medical Drive, Singapore.

^*Corresponding author:

Food Science and Technology Programme

Department of Chemistry

National University of Singapore

3 Science Drive 3, Singapore 117543

Tel.: 65-6516-8821

Fax: 65-6775-7895

E-mail: chmhdj@nus.edu.sg

Abstract

A fluorescent assay for simultaneous screening of myeloperoxidase (MPO) inhibitors and hypochlorous acid (HOCl) scavengers was developed using quantum dots (QDs) as a selective HOCl probe. HL60 cells were differentiated into neutrophil phenotype and used for HOCl generation in this assay. The fluorescence of QDs was specifically quenched by HOCl generated from the neutrophil-like cells induced with phorbol 12-myristate 13-acetate (PMA) or hydrogen peroxide (H₂O₂). Both MPO inhibitors (e.g. resveratrol) and HOCl scavengers (methionine and vitamin C) tested in this assay could inhibit the QDs fluorescence quenching but MPO inhibitors showed a more obvious dose response relationship than HOCl scavengers. A microplate assay under the same conditions using 2,7-dichlorofluorescin diacetate (DCFH-DA), a commonly used reactive oxidative species (ROS) probe, was also performed to make a comparison with QDs based assay. The results indicated superior HOCl specificity of QDs over DCFH-DA and necessity of using ROS probes with different selectivity for a comprehensive evaluation of antioxidant efficiency in cellular systems. This QDs based microplate assay has a potential to be used in cell line-based high throughput screening for HOCl scavengers or MPO inhibitors with therapeutic importance in controlling inflammation.

Keywords: Quantum dots; Myeloperoxidase; HL60 cells; Hypochlorous acid; Inhibitor.

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