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

by Jinfang Liao, Zhenjun Diwu, Jixiang Liu, Qin Zhao, Ruogu Peng, Zhen Luo

Introduction

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

Cell Culture

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

Total ROS Assay

Total ROS indicator: Amplite ROS Green.
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

O₂^•- used was MitoROS™ 580.
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

NO indicator used was Nitrixyte™ Orange
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

ONOO^- indicator used was DAX-J2 PON Green
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

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.

Table 1. Fluorescence properties of ROS and RNS reagents.

Probe ▲ ▼	Target ▲ ▼	Ex/Em (nm) ▲ ▼	Flow Cytometer Channel ▲ ▼	Microplate Reader Setting Ex/Em (nm) ▲ ▼	Cutoff (nm) ▲ ▼
Amplite® ROS Green	Total ROS	492/520	FITC	490/525	515
DAX-J2™ PON Green	Peroxynitrite	502/535	FITC	490/530	515
Nitrixyte™ Orange	Nitric Oxide	545/576	PE	540/590	570
MitoROS™ 580	Superoxide	540/590	PE	540/590	570

Flow Cytometric Measurment of Total ROS

(A)

(B)

(A) Flow cytometric analysis of total ROS generation in Jurkat cells upon TBHP treatment. (B) Changes in fluorescence intensity corresponding to total ROS generation in the curcumin treated and control Jurkat cells. Fluorescence intensity was measured using a BD FACSCalibur™ cell analyzer.

Flow Cytometric Measurment of Superoxide

(A)

(B)

(A) Flow cytometric analysis of O₂^•- generation in Jurkat cells upon AMA treatment. (B) Changes in fluorescence intensity corresponding to O₂^•- generation in the curcumin treated and control Jurkat cells. Fluorescence intensity was measured using BD FACSCalibur.

Flow Cytometric Measurment of Nitric Oxide

(A) Flow cytometric analysis of NO generation in Jurkat cells upon NONOate treatment. (B) Changes in fluorescence intensity corresponding to NO generation in the curcumin treated and control Jurkat cells. Fluorescence intensity was measured using BD FACSCalibur.

Flow Cytometric Measurment of Peroxynitrite

Flow cytometric analysis of ONOO^- generation in Jurkat cells upon SIN-1 treatment for 1 hour and 16 hours. (B) Changes in fluorescence intensity corresponding to ONOO^- generation in the curcumin treated and control Jurkat cells. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer.