Flow Cytometric Analysis of Intracellular ROS and RNS Production and Curcumin Inhibition

Introduction

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Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important biological regulators involved in cell damages and health problems. Curcumin, a naturally occurring phenolic compound, has long been recognized as a promising anticancer medicine because it can effectively inhibit ROS generation. However, ROS is a mixture of oxidative species, and the effects of curcumin on different oxidative molecules are unclear. Moreover, ROS analysis via flow cytometry is challenging due to the lack of specific probes for selective detection of ROS and RNS species. To address these issues, we have developed a few novel ROS and RNS probes with high selectivity targeting different ROS and RNS species.

Materials and Method

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Cell Culture

1. Human T lymphocyte Jurkat cells were grown at 37 °C in RPMI 1640.

Total ROS Assay

1. Total ROS indicator: Amplite ROS Green.
2. Jurkat cells pre-incubated with Amplite ROS Green for 1 hour were then treated with 100 µM TBHP (tert-Butyl hydroperoxide) in growth medium at 37 °C for 30 minutes to induce exogenous ROS.

Superoxide (O₂^•-) Assay

1. O₂^•- used was MitoROS™ 580.
2. Jurkat cells were treated with 50 µM Antimycin A (AMA) at 37 °C for 30 minutes to induce endogenous O₂^•-, then incubated with MitoROS™ 580 for 1 hour.

Nitric oxide (NO) Assay

1. NO indicator used was Nitrixyte™ Orange
2. Jurkat cells were incubated with Nitrixyte Orange for 30 minutes. After washing with Hanks and 10 mM HEPES buffer, cells were further treated with 1 mM DEA NONOate in Hanks and 10 mM HEPES buffer at 37 °C for an additional 30 minutes to induce exogenous NO.

Peroxynitrite (ONOO^-) Assay

1. ONOO^- indicator used was DAX-J2 PON Green
2. Jurkat cells were incubated with DAX-J2 PON Green for 1 hour and then treated with 200 µM SIN-1 in growth medium at 37 °C for 1-16 hours to induce endogenous ONOO^-.

Curcumin Treatment

1. urkat cells were pre-treated with curcumin in growth medium at 37 °C for 4 hours, then pelleted by centrifugation. Cells were resuspended in growth medium or desired buffer and processed to total ROS, O₂^•-, NO or ONOO^- assay as described above.

Flow Cytometric Measurment of Total ROS

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Flow Cytometric Measurment of Superoxide

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Flow Cytometric Measurment of Nitric Oxide

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Flow Cytometric Measurment of Peroxynitrite

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