Monitoring viability using cellular reducing environment
The alamarBlue HS and alamarBlue Cell Viability Reagents are ready-to-use resazurin-based reagents that function as cell health indicators by using the reducing power of living cells to quantitatively measure viability. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent.
This protocol can be used for:
- Identifying live mammalian, bacterial, plant, fungal cells using a microplate reader
- Higher cell densities
- Longer incubation times
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:
- alamarBlue Cell Viability Reagent
- alamarBlue 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 alarmaBlue HS or alarmaBlue reagent to microplate wells (see recommended volumes) |
 3. Incubate at 37ºC for 1–4 hours |
 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 alamarBlue HS or alamarBlue reagent can be replaced with complete media and returned to the incubator. The cells will continue to proliferate normally.
Spectral information and storage
| alamarBlue HS or alamarBlue 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. alamarBlue HS and alamarBlue reagent recommended volumes.
Both alamarBlue Cell Viability Reagents are supplied as 10X solutions. Add alamarBlue reagent directly to cells in culture medium. See below for example volumes:
| Format | Volume of cells + medium | Volume of alamarBlue HS or alamarBlue reagent |
|---|---|---|
| Cuvette | 900 μL | 100 μL |
| 96-well plate | 90 μL | 10 μL |
| 384-well plate | 36 μL | 4 μL |