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Resazurin Cytotoxicity

Background

Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a mildly fluorescent blue dye that, upon reduction, becomes the highly fluorescent pink resorufin. The irreversible reduction of resazurin to resorufin is proportional to aerobic respiration. This makes it useful for detecting the viability of bacteria and mammalian cells by their redox capabilities.

Procedure (for 384-well plate)

  1. Make a 500 uM resazurin stock solution in PBS (stored at -20 °C or 4 °C).

    Warning

    If storing at -20 °C, reduce the number of freeze/thaw cycles — no more than 10.

  2. Filter-sterilize the solution through a 0.2 um filter into a sterile, light-protected container.

  3. Create a working solution of 10 uM from the resazurin stock solution.
  4. Prepare 384-well plates with cells and compound to have a final well volume of around 25 uL/well. (See Tissue Culture.)
  5. Incubate cells at 37 °C for the desired amount of time.
  6. Add 10 uL/well of the 10 uM resazurin solution to cells.
  7. Incubate plates 1–4 hours at 37 °C.
  8. Read fluorescence at 560 nm excitation and 590 nm emission.