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- Specifically designed to measure Superoxide production in live cells
- Compatible with major components of tissue culture media (phenol red, FBS and BSA)
- Validated on microscopy, flow cytometry, and microplates
Enzo Life Sciences ROS-ID® Total ROS/Superoxide detection kit includes dyes to measure Reactive Oxygen Species (ROS) and Superoxide production in live cells. The non-fluorescent, cell-permeable Oxidative Stress Detection Reagent (Green, Ex/Em 490/525 nm) reacts directly with a wide range of reactive species (hydrogen peroxide, peroxynitrite and hydroxyl radicals) yielding a green fluorescent product. The Superoxide Detection Reagent (Orange, Ex/Em 550/620 nm) is a cell-permeable probe that reacts specifically with superoxide, generating an orange fluorescent product. Pyocyanin (ROS inducer) and NAC (ROS inhibitor) are also provided with the kit.
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Figure 6. ROS-ID® Total ROS/Superoxide detection Kit and a set of ROS scavengers/inhibitors were used to profile ROS production in HeLa cells treated with antimycin A, (AMA, specific superoxide inducer), t-butyl-peroxide (TBHP, specific peroxide inducer), and pyocyanin (general ROS inducer). Similar results were obtained using U-2 OS and CHO K1 cells.
Figure 4. Profiling of ROS formation by flow cytometry in Hela cells. Data represents % positive following treatment with Pyocyanin (ROS/SO inducer), TBHP (ROS inducer), and AMA (superoxide inducer). Green columns indicate % positive for ROS and orange columns represent % positive for superoxide.
Figure 1. Profiling of reactive oxygen species formation by fluorescence microscopy was achieved in HeLa cells loaded with ROS/Superoxide detection reagents and treated with pyocyanin. General oxidative stress levels were monitored in the green channel, while superoxide production was detected in the orange channel. Pretreatment with NAC, a general ROS inhibitor, prevents formation of ROS.
Figure 3. Flow cytometry histograms of ROS inhibition by N-acetyl-L-cysteine (NAC, ROS inhibitor). Jurkat cells were pretreated with 5 mM (pink, left) or 10 mM (pink, right), or without NAC (green, positive control) for one hour. Cells were then incubated with 100 µM pyocyanin (ROS inducer) and stained with 1 µM oxidative stress detection reagent. Untreated cells (blue) were used as a negative control. The numbers in the upper right corners indicate the percentage of median fluorescence intensity of NAC-treated cells as compared with positive control cells.
Figure 5. (Top) HeLa Cells Loaded with ROS/RNS 3-Plex Detection Reagent for 2 h, 37°C and Induced for 20 min, 37°C.
Column 1: Control
Column 2: Pyocyanin, 0.1 mM
Column 3: L-Arginine, 1mM
Column 4: Pyo/Arg
(Bottom) Pretreatment with 5 mM NAC
Column 1: Control
Column 2: L-Arginine, 1mM
Column 3: Pyocyanin, 0.1 mM
Column 4: Pyo/Arg
Specific profiling of reactive oxygen/nitrogen species formation by fluorescence microscopy in HeLa cells loaded with dyes from ROS-ID® ROS/RNS Detection Kit (ENZ-51001). (Top) – Combined treatment of Pyocyanin and L-Arg generates peroxynitrite (green fluorescence) due to the reaction of NO with superoxide, bur little NO (red fluorescence). (Bottom) – Pretreatment with NAC inhibits peroxynitrite and superoxide formation, but not NO.
Figure 2. Jurkat cells were induced with 100 µM pyocyanin (general ROS inducer, panel B), 200 µM antimycin A (superoxide inducer, panel C) or 1 µM of t-butyl-hydroperoxide (peroxide inducer, panel D), stained with two color ROS Detection Kit and analyzed using flow cytometry. Untreated cells (panel A) were used as a control. Cell debris were ungated and compensation was performed using single stained pyocyanin-treated samples. Red numbers reflect the percentage of the cells in each quadrant.
ROS-ID® Total ROS/Superoxide detection kit Kit box image
| Regulatory Status |
RUO – Research Use Only |
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