Cell-based viability assays are important to determine whether cells are alive or dead. Such assays are widely used for investigating cell response to a given agent, drug or to establish relative cytotoxicity of chemicals.
Living cells maintain a reducing environment within their cytoplasm and mitochondria, in which resazurin (blue and non-fluorescent) is reduced by dehydrogenase enzymes to form the red fluorescent dye resorufin. The amount of resorufin can be monitored by measuring fluorescence or absorbance, which is proportional to the number of living cells in the sample. Depending on the cell types, the resazurin assay can be used to detect as few as 40 cells with reproducible and sensitive signal.
Figure 1. The principle of resazurin cell viability assay.
- Sensitive: requires as few as 80 cells for reproducible results
- Simple: cell lysis, cell fixation, and cell permeabilization are not required
- Kinetic monitoring: non-toxic to cells, thus allowing long-term dynamic monitoring
- Safe: non-toxic, non-radioactive, non-carcinogenic, requires only simple disposal
- Versatile: successfully used in a wide range of cell types, including bacteria, protozoa, yeast, fungi, and cultured mammalian cells
- 96-well plate
- Cell culture reagents
- Mitochondrial viability dye
- A fluorescent microplate reader
- Multi and single channel pipettes
The protocol below is described for a 96-well plate. If the assay is performed on a 384-well plate, adjusting volumes accordingly. This assay has been optimized for adherent and suspension cells.
- Seed cells into a 96-well plate with 100 μL/well. The optimal cell seeding density is dependent on cell type and duration of experimental time course.
Note: Ensure that there will be at least one well with 100 μL of cell culture medium without cells to use as a background control.
- After cells have reached the desired density, add 10 μL of resazurin solution to the medium in each well and mix thoroughly.
- Incubate the plate at 37°C for 4 hours.
Note: Signals from the same plate can be read at multiple time points to determine the optimal incubation time for different cell types and densities.
- Read the plate by using a fluorescence microplate reader with excitation at 570 nm and emission at 590 nm.
Note: The excitation and emission spectra of resorufin are fairly broad, excitation filters between 530-570 nm and emission filters between 580-620 nm can be used.
- For fluorescence-based detection, subtract fluorescence at 590 nm from the background control (culture medium without cells) from each cell sample.
- Riss T.L. et al.; Cell viability assays. Assay Guidance Manual, 2013.
- Borra R.C. et al.; A simple method to measure cell viability in proliferation and cytotoxicity assays. Braz Oral Res, 2009, 23 (3): 255-262.
Cell Line Testing and Assays: