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Imaging examples and protocols for OxiORANGE™.

Detection of hROS generation by HeLa cells.

Cell staining protocol
  1. Add 100 μL of dimethylformamide (DMF) to one vial of OxiORANGE™ (100 nmol) to make 1 mM stock solution.
  2. Dilute the stock solution with HBSS or cell culture medium to make 1 µM cell staining solution.
    ※ Optimize the concentration and incubation time is required depending on the cells and culture condition. In our lab., staining with 1 µM solution for 20 minutes at 37℃ gave good results for HeLa, RAW264.7, RBL-2H3, and HL60 cells.
  1. Remove culture media, and rinse cells with HBSS.
  2. Add the cell staining solution and incubate for 20 minutes at 37℃, 5% CO2.
  3. Rinse cells with HBSS, for two times..
  4. Replace with appropriate observation medium.

    ※ We recommend to use culture medium without phenol red for HeLa cells, because starvation of HeLa cells enhances ROS production.
    Stimulate cells by the addition of 500 µM H2O2 to induce ROS production, and start time-lapse imaging.

    ※ In our test, fluorescence of OxiORANGE™ was detected after a few minutes. After 15 minutes, strong fluorescence was observed.

hROS generation detected with OxiORANGE™ in HeLa cells.

hROS generation detected with OxiORANGE™ in HeLa cells.
※ Observed using NIKON ECLIPSE Ti, microscope (PlanFluor 40×0.75), and Hamamatsu ORCA-R2 camera.

Addition of antioxidant (N-acetyl-cysteine, NAC) reduces ROS production (right).

Find Youtube timelapse video.

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