alamarBlue Assays for Cell Viability Protocol, for Microplates

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.

Learn more about alamarBlue Cell Viability Assays

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
  - 25 mL (Cat. No. DAL1025)
  - 100 mL (Cat. No. DAL1100)
- alamarBlue HS Cell Viability Reagent
  - 25 mL (Cat. No. A50100)
  - 100 mL (Cat. No. A50101)
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 alamarBlue HS or alamarBlue 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