Intracellular Total ROS Activity Assays

In this issue

Maleimide Assays
Glycerol Assays
Glycerol 3-Phosphate Assays
Acetylcholinesterase Assays
Nucleic Acid Quantification
Multicolor Intracellular Calcium Detection Probes
Intracellular Nitric Oxide (NO) Assays
Intracellular Total ROS Activity Assays
iFluor™ 700 Dyes
HRP Antibody Labeling Using Buccutite™ Crosslinking Technology
6-JOE SE

AssayWise Letters 2014, Vol. 3(2)
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Detection of intracellular ROS in Jurkat cells upon TBHP treatment using Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit (Cat# 22904). Cells were incubated with Amplite^® ROS Green at 37°C for 1 hour. Cells were then treated with (Red) or without (Blue) 100 µM TBHP at 37°C for 30 minutes. The fluorescence signal was monitored using a flow cytometer (BD FACSCalibur™) in FL1 channel.

Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen (such as superoxide, hydroxyl radical, singlet oxygen and peroxides). ROS is highly reactive due to the presence of unpaired valence shell electrons. ROS forms as a natural byproduct of the normal metabolism of oxygen and plays important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. It may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. ROS is also generated by exogenous sources such as ionizing radiation. Under the conditions of oxidative stress, greatly increased production of ROS results in subsequent alteration of membrane lipids, proteins and nucleic acids. Oxidative damage of these biomolecules is associated with aging as well as with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemic reperfusion injury, and neurodegenerative disorders.

Amplite^® Fluorimetric Intracellular Total ROS Activity Assay Kits provide a sensitive, one-step fluorimetric assay to detect intracellular ROS in live cells. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using either a fluorescence microplate reader or a fluorescence microscope. Amplite^® Fluorimetric Intracellular Total ROS Activity Assay Kits (Cat# 22900, 22901, 22902 and 22903) are in an optimized "mix and read" assay format that is compatible with HTS liquid handling instruments. Kit 22904 is optimized for flow cytometry applications, its signal can be detected at Ex/Em = 490/520 nm (FL1 channel).

Left: Detection of ROS in Jurkat cells using Amplite^® Fluorimetric Intracellular Total ROS Activity Assay Kit (Cat# 22900). Jurkat cells were seeded on the same day at 300,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The ROS assay loading solution (100 µL/well) was added and incubated in a 5% CO₂, 37°C incubator for 1 hour. And then the cells were treated with 1 mM, 0.1 mM or without H₂O₂ for 30 minutes. Right: Detection of ROS in Jurkat cells using Amplite^® Fluorimetric Intracellular Total ROS Activity Assay Kit (Cat# 22901). Jurkat cells were seeded on the same day at 300,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The ROS assay loading solution (100 µL/well) was added and incubated in a 5% CO₂, 37 °C incubator for 1 hour. And then the cells were treated with or without 1mM H₂O₂ for 2 hours.

Images of HeLa cells stained with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit (Cat# 22902) in a Costar 96-well black wall/clear bottom plate. Top: Untreated control cells. Bottom: Cells treated with 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes before staining.

Images of HeLa cells stained with Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit (Cat# 22903) in a Costar 96-well black wall/clear bottom plate. Top: Untreated control cells. Bottom: Cells treated with 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes before staining.

Left: Detection of ROS in HeLa cells. HeLa cells were seeded overnight at 15,000 cells/90 µL/well in a Costar 96-well black wall/clear bottom plate. The cells were untreated (control) or treated with 1 mM H₂O₂ or 100 µM tert-butyl hydroperoxide (TBHP) for 30 minutes at 37 °C. The ROS Brite™ 570 (Cat# 16000) assay solution (100 µL/well) was added and incubated in a 5% CO₂, 37 °C incubator for 1 hour. The fluorescence signal was monitored at Ex/Em = 540/570 nm (cut off = 550 nm) with bottom read mode using FlexStation^® (Molecular Devices). Center: Detection of ROS in Jurkat cells. Jurkat cells were treated without (Blue) or with 100 µM tert-butyl hydroperoxide (TBHP) (Red) for 30 minutes at 37 °C, and then loaded with ROS Brite™ 570 (Cat# 16000) in a 5% CO₂, 37 °C incubator for 1 hour. The fluorescence intensities were measured with a FACSCalibur™ flow cytometer using FL2 channel. Right: Detection of ROS in Jurkat cells. Jurkat cells were treated without (Blue) or with 100 µM tert-butyl hydroperoxide (TBHP) (Red) for 30 minutes at 37 °C, and then loaded with ROS Brite™ 670 (Cat# 16002) in a 5% CO₂, 37 °C incubator for 1 hour. The fluorescence intensities were measured with a FACSCalibur™ flow cytometer using FL2 channel.

Table 1. Intracellular Total ROS Activity Assay Kits

Cat No. ▲ ▼	Product Name ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Unit Size ▲ ▼
22903	Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitDeep Red Fluorescence	658	675	200 Tests
22904	Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitOptimized for Flow Cytometry	490	520	100 Tests
22902	Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitOrange Fluorescence	556	566	200 Tests
22901	Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitRed Fluorescence	520	605	200 Tests
22900	Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay KitGreen Fluorescence	494	520	200 Tests