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.

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)
    • 25 mL (Cat. No. A50100)
    • 100 mL (Cat. No. A50101)

    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