PrestoBlue Cell Viability Reagent for Microplates Protocol

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.

Learn more about PrestoBlue Cell Viability Assays

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:

Cells growing in culture
One of the following:
- PrestoBlue Cell Viability Reagent
  - 25 mL (Cat. No. A13261)
  - 100 mL (Cat. No. A13262)
- PrestoBlue HS Cell Viability Reagent
  - 25 mL (Cat. No. P50200)
  - 100 mL (Cat. No. P50201)
Phosphate-buffered saline (PBS) (Cat. No. 14190-144)
Fluorescence or absorbance microplate reader

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	560/590 nm
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

PrestoBlue Assays for Cell Viability Protocol, for Microplates