image of a PrestoBlue reagent bottles

Reducing environment—sensing cell viability assay

The PrestoBlue HS (high sensitivity) and PrestoBlue reagents contain resazurin, which is a non-toxic, cell-permeant compound that is blue in color and virtually non-fluorescent in solution. When added to media, the 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.

The PrestoBlue HS Cell Viability Reagent uses the same proprietary formulation as the PrestoBlue Cell Viability Reagent, except that highly purified resazurin is used for the HS formulation. The highly purified resazurin used for PrestoBlue HS results in a reagent will >50% decrease in background fluorescence and a >100% in the signal to background ratio. The PrestoBlue HS Cell Viability Reagent is a complete add-and-read, non-toxic reagent that does not require cell lysis. Since no lysis is required, the diluted PrestoBlue HS solution can be removed, replaced with complete growth media, and the cells can be cultured further.

Unlike other resazurin-based reagents, the PrestoBlue HS and PrestoBlue Cell Viability Reagents have been formulated with a propriety buffering system; resulting in a reagent with a physiological pH range optimal for the quick determination of mammalian cell viability. Incubation times using PrestoBlue HS and PrestoBlue are significantly shorter than with other resazurin-based cell viability reagents. Cell viability can be detected with a short 10 minute incubation using the PrestoBlue Cell Viability reagents.

This protocol can be used for:

  • Identifying 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
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 reagents 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
96-well plate 90 μL 10 μL
384-well plate 36 μL 4 μL
1,536-well plate 5 μL 3 μL