image of a PrestoBlue reagent bottles

Fast determination of mammalian cell viability by sensing the reducing environment present in live cells

The PrestoBlue reagents, PrestoBlue HS (high sensitivity) and PrestoBlue, contain resazurin and a propriety buffering system. When added to media, the PrestoBlue reagents are rapidly taken up by cells. The reducing environment within viable cells converts the non-toxic resazurin in the PrestoBlue reagent to an intensely red-fluorescent dye. This change can be detected by measuring fluorescence or absorbance.

This protocol can be used for:

  • Rapid identification of live mammalian 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 either PrestoBlue HS or 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

Note regarding subsequent culture
After viability determination, the diluted PrestoBlue HS or PrestoBlue reagent can be replaced with complete media and returned to the incubator. The cells will continue to proliferate normally.

Spectral information and storage
  PrestoBlue HS and PrestoBlue Cell Viability Reagents
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 HS and PrestoBlue reagent recommended volumes.

Both PrestoBlue Cell Viability Reagents are supplied as 10X solutions. Add PrestoBlue reagent directly to cells in culture medium. See below for example volumes:

Format Volume of cells + medium Volume of PrestoBlue HS or PrestoBlue reagent
Cuvette 900 μL 100 μL
96-well plate 90 μL 10 μL
384-well plate 36 μL 4 μL
1,536-well plate 5 μL 3 μL