ROS-ID^(R) Total ROS/Superoxide detection kit  

ROS-ID® Total ROS/Superoxide detection kit Flow Cytometry％％Figure 2. Jurkat cells were induced with 100 &microM pyocyanin (general ROS inducer, panel B), 200 &microM antimycin A (superoxide inducer, panel C) or 1 &microM 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 double-chart％％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^(R) 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.

ROS-ID® Total ROS/Superoxide detection kit Flow Cytometry％％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.

ROS-ID® Total ROS/Superoxide detection kit Fig1％％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.

ROS-ID® Total ROS/Superoxide detection kit Flow Cytometry％％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.

ROS-ID® Total ROS/Superoxide detection kit doublegraphs％％Figure 6. ROS-ID^(R) 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.