alamarBlue Cell Viability Assay

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:

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 to 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 (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. 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