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:
- Cells growing in culture
- One of the following:
- PrestoBlue Cell Viability Reagent
- PrestoBlue HS Cell Viability Reagent
- 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.
| 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 |