For more information refer to:
https://tools.thermofisher.com/content/sfs/manuals/Image_iT_Hypoxia_Reagent_UG.pdf

Prepare stock solutions:

Image-iT® Hypoxia Reagent is provided as a lyophilized powder. To make 1 mM stock solution, dissolve the hyophilized powder in 1.40 mL of DMSO. This stock solution can be stored at ≤−20°C for 6 months, or 2°C to 8°C for up to one week. For best results, mix well when preparing the stock solution and avoid the freeze-thaw cycles.

  1. Plate cells at the recommended density on a glass bottom dish and incubate them overnight at 37°C overnight in a CO2 incubator. For better results, higher cellular density is recommended.
  2. Prepare a 1 mM stock solution of the Image-iT® Hypoxia Reagent in DMSO. Add the reagent to the cells at a final concentration of 5–10 µM and incubate for 15 to 30 minutes.
  3. Place the cells in a hypoxia chamber set to the desired oxygen concentration and attached to a fluorescence microscope, and incubate the cells.
  4. Image the cells on the fluorescence microscope with excitation/emission of 490/610 nm. Note: You can use the standard TRITC filter set for imaging; however, we recommend using FITC excitation and Texas Red® emission filters for best results.