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