Introduction

Reactive oxygen species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. Recent work has demonstrated that ROS have a role in cell signaling, including apoptosis, gene expression, and the activation of cell signaling cascades (1).

NADPH oxidase is a well known producer of ROS moieties (1). Its activity is controlled by a complex regulatory system that involves the G protein Rac (2). In resting cells, a membrane-embedded heterodimer of two polypeptides (p22phox and gp91phox)—which also contains two heme groups as well as a FAD group—enables the transfer of electrons from cytosolic NADPH across the membrane to molecular oxygen without NADPH oxidase activity (1). It is believed that the charge compensation occurs when gp91phox polypeptide also acts as a proton pump. Upon stimulation, a number of polypeptides (p47phox, p67phox, and p40phox) translocates to the inner face of the plasma membrane to form a fully active enzyme complex that contains NADPH oxidase activity (Figure 1).

Figure 1. Schematic illustration of the activation of NADPH oxidase. (Click to enlarge)

Part of the active complex is the G protein Rac, which, upon stimulation, dissociates from GDI, binds GTP and associates with the membrane (2). The assembled complex then catalyzes the formation of hydrogen peroxide (H₂O₂) from oxygen and hydrogen ions (Figure 1).

DCF reactions

In order to observe the formation of reactive oxygen species a fluorescent detector is employed. The acetate ester form of 2′,7′ dichlorodihydrofluorescein diacetate (H₂DCFDA-AM) is a membrane-permeant molecule that passes through the cell membrane. Once inside the cell, cellular esterases act on the molecule to form the non-fluorescent moiety.

H₂DCFDA is ionic in nature and therefore trapped inside the cell. Oxidation of H₂DCFDA by ROS converts the molecule to 2′,7′ dichlorodihydrofluorescein (DCF), which is highly fluorescent. Upon stimulation, the resultant production of ROS causes an increase in fluorescence signal over time.

Materials and Methods

Primary mesothelioma cells were cultivated in DMEM (10% FCS). Cell lines were trypsinized, counted and resuspended in fresh media at a density of 30,000 cells/mL. Using a MicroFlo peristaltic pump dispenser (BioTek Instruments), cells were seeded into Corning 24-well plates (Catalog no. 3337; Corning, NY, USA) at 37°C.

The following day, the media was replaced with serum-free DMEM and placed back in the incubator overnight.

Cells were loaded with dye by replacing media with fresh phenol red–free DMEM containing 5 µM H₂DCFDA-AM for 30 minutes at 37°C, in a 5% CO₂ environment. After loading, unincorporated dye was removed by washing two times with fresh media. After washing, media containing experimental conditions was added and the fluorescence of the wells measured kinetically.

Fluorescence measurements were made using a Synergy HT Microplate Reader (BioTek Instruments) set to 37°C. Measurements were made using a 485/20 nm excitation and a 528/20 nm emission filter pair and a PMT sensitivity setting of 55. Readings were made from the bottom every 30 seconds for a total of 45 minutes.

Results and Discussion

Figure 2 demonstrates the ability of the Synergy HT to detect cell stimulation using DCF fluorescence under the defined experimental conditions. Phorbol 12-myristate 13-acetate (PMA) and serum are known to stimulate cell growth in quiescent cells. Both of these agents induce the formation of DCF fluorescence over time, while unstimulated cells do not show an increase. Glutathione oxidase, which generates ROS without cellular stimulation, is used as a positive control. As demonstrated in Figure 2, agents that are known to stimulate cellular proliferation also induce the formation of ROS, which in turn leads to the production of DCF fluorescence. The increase in fluorescence is not limited by the availability of substrate. Wells with glutathione oxidase, which generates the ROS, and H₂O₂, as a byproduct of the oxidation of glutathione, demonstrate a continuous increase in fluorescence throughout the experiment. This indicates that DCF substrate is not the limiting agent in the assay.

Figure 2. DCF fluorescence in stimulated cells measured over time. (Click to enlarge)

Figure 3 demonstrates endpoint fluorescence 30 minutes after the initiation of treatment. Both PMA and serum demonstrate a marked increase in fluorescence when compared to the unstimulated control. When the background fluorescence, depicted by the no DCF control is subtracted, the increase by 0.25% serum represents a 3-fold increase, while the 1 µM PMA stimulation was nearly 5-fold.

Figure 3. PMA and serum induction of H₂O₂ as measured by endpoint fluorescence after 30 min. (Click to enlarge)

These data demonstrate that the Synergy HT can be used to detect the formation of intracellular ROS as a result of the stimulation of primary mesothelioma cells. Increases in DCF fluorescence measured by the Synergy HT follow expected cellular stimulation patterns with differences in signal from background being evident almost immediately.

There are a number of different compounds that can be used to assess ROS production. However, many of these compounds are not necessarily suitable for measurements of cytoplasmic ROS. The compound DCF does pass through intact cell membranes and interacts with ROS species such as H₂O₂ to form a fluorescent moiety, but is not necessarily specific for this molecule. DCF is actually more reactive with radical ions such as hydroxyl radical (HO•) or peroxyl radical (ROO•) than H₂O₂. The development of H₂O₂-specific probes such as Peroxyl Green 1 will allow for specific analysis of the formation of H₂O₂ (3).

The Synergy family of readers provides an excellent platform for making ROS determinations in stimulated cells. The readers provide highly sensitive fluorescence detection through the use of dedicated optics in conjunction with deep blocking bandpass filters and a photomultiplier tube (PMT). In these experiments tight temperature control is maintained at 37°C, but the reader is capable of temperatures up to 50°C. Gen5 data reduction software (BioTek Instruments) was used to control reader function, as well as collect and plot the data. This software package allows for most common data reduction transformations, curve fitting, and data analysis.