image of a PrestoBlue reagent bottles

Reducing environment—sensing cell viability assay

PrestoBlue® reagent contains a cell-permeant compound that is blue in color and virtually nonfluorescent in solution. When added to media, PrestoBlue® reagent is rapidly taken up by cells. The reducing environment within viable cells converts PrestoBlue® reagent to an intensely red-fluorescent dye. This change can be detected by measuring fluorescence or absorbance.

This protocol can be used for:

  • Identifying live cells using a microplate reader

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

Assay protocol

1. Add cells in appropriate medium to microplate wells
2. Add PrestoBlue® reagent to microplate wells (see recommended volumes)
3. Incubate at 37ºC for 10 minutes
4. Read fluorescence or absorbance (signal is stable for 7 hours)
5. Plot a curve of relative fluorescence units vs. drug concentration to generate quantitative results
Spectral information and storage
  PrestoBlue® Cell Viability Reagent
Excitation/Emission (in nm) 560/590
Absorbance <570 nm (use 600 nm reference wavelength)
Storage conditions 4°C


Protocol tips

  • Correct for background fluorescence by including control wells containing only cell culture medium (no cells) on each plate
  • Fluorescence is more sensitive than absorbance and is the preferred detection method
  • Bottom-read is more sensitive than top-read

Table 1. PrestoBlue® reagent recommended volumes.

PrestoBlue® Cell Viability Reagent is supplied as a 10X solution. Add PrestoBlue®  reagent directly to cells in culture medium. See below for example volumes:

Format Volume of cells + medium Volume of PrestoBlue® reagent
96-well plate 90 μL 10 μL
384-well plate 36 μL 4 μL
1,536-well plate 5 μL 3 μL