Microplate assays provide information on entire cell populations rather than tracking the behavior of individual cells. We offer microplate assays for whole cells and assays performed on disrupted cells or cell lysates. Metabolic activity is commonly used as a viability indicator, but for some applications it can be important to assess viability independent of metabolic state, and appropriate assays are listed below. In some cases, cells are required for additional downstream functional analysis, and alamarBlue® is an excellent nontoxic indicator of viability.
Microplate Assays for Cell Viability
Measuring cellular reduction potential
The inherent reducing power of live cells serves as an indicator of metabolic activity and thus cell viability; when cellular reduction potential is measured in a population, the signal is proportional to the number of live cells. Some established assays such as those using MTT use that reducing power to generate a strongly colored reporter, formazan. Similarly, the redox indicator in our alamarBlue® and PrestoBlue® cell viability assays (resazurin) is converted to the fluorescent and colorimetric reporter molecule resorufin in metabolically active cells, and it serves as a very reliable and sensitive viability/cytotoxicity indicator.
We’ve developed these alamarBlue® and PrestoBlue® assays to be simple and convenient by providing the reagents in a proprietary stabilizing formulation with a “mix, incubate, and read” protocol that is scalable from single wells to high-throughput screening (HTS).
Measuring membrane permeability
The loss of plasma membrane integrity is a key marker of cell death that is regularly exploited by tools that measure viability and cytotoxicity. Cells with compromised plasma membranes allow the influx of cell-impermeant DNA-binding dyes that fluoresce only when bound to DNA. Thus, DNA staining can be used as a cytotoxicity indicator that is independent of metabolic state. Cytotoxic compounds can damage cells through a range of mechanisms. Cell viability, membrane integrity, and DNA content are among the most specific and sensitive parameters for measuring cytotoxicity, and these dyes provide excellent sensitivity and accuracy.
A richer result is generated when DNA binding is combined with measurements of other parameters. Probes for esterase activity or cellular reduction potential stain live cells and provide measures of viability. A cell-permeant DNA stain will also stain cells with intact plasma membranes and serve as a marker for viable cells.
Assays for microorganisms
Microbial viability can be determined using imaging assays that measure membrane permeability. A combination of membrane-permeant and -impermeant DNA dyes can be used to stain intact cells green and dead cells red. For bacteria, live cell determination can also be combined with fluorescent Gram staining.
For yeast cells, a membrane stain is used to detect both live and dead cells with a blue signal. In viable cells, the vacuole stains orange/red, providing a two-color assay.
Multiplexing CellEvent™ and PrestoBlue® reagents using high-throughput screening (HTS). CHO-K1 cells were plated at 5,000 cells/well in a 384-well plate in the presence of a dilution series of staurosporine, in phenol red–free complete media. The plate was incubated for 19 hr at 37°C/5% CO2. PrestoBlue® reagent and CellEvent™ reagent were added directly to the wells, and the plate was incubated for 30 min at 37°C/5% CO2 prior fluorescence measurement. Florescence signal was detected on a Tecan Safire2 (bottom read) with settings of Ex/Em=500/530 nm (bandwidths of 7 nm) for the CellEvent™ reagent and Ex/Em=560/590 nm (bandwidths of 10 nm) for the PrestoBlue® reagent.
|alamarBlue®||PrestoBlue® Cell Viability Reagent|
|Target||Metabolic activity||Metabolic activity|
|Sample||Whole cells or lysate||Whole cells|
|Usage||Measures cellular reduction potential||Measures cellular reduction potential|
|Components||Bulk reagent||Bulk reagent|
|Format||25 mL||25 mL|
|LIVE/DEAD® Viability/Cytotoxicity Kit, for mammalian cells||Vybrant® Cytotoxicity Assay Kit (G6PD release Assay)||Image-IT® DEAD Green™ Viability Stain||HCS LIVE/DEAD® Green Kit|
|Target||Mammalian cell viability||Mammalian Cell Viability||Mammalian Cell Viability||Mammalian Cell Viability|
|Reporter||Calcein AM/ethidium homodimer-1||Resazurin||Image-IT® DEAD Green™||Image-IT® DEAD Green™, NuclearMask™ Deep Red or Hoechst 33342|
|Ex/Em (nm)||488/535 & 528/617||540 / 590||488/515||488/515, 638/686, or 350/461|
|Sample||Adherent or suspended cells||Cell suspension and surrounding media||Adherent or suspended cells||Adherent cells|
|Usage||Quick and easy two-color assay based on plasma membrane integrity and esterase activity||Quick and easy assay based on G6PD release from damaged cells||Quick and easy assay based on plasma membrane integrity||Two-color assay based on plasma membrane integrity|
|Components||Bulk dye solutions||Complete assay||Bulk dye solution||Complete assay|
|Format||1 kit, 10 microplates||1 kit, 10 microplates||1 vial, 25 plates||1 kit, 2 plates|
|LIVE/DEAD® BacLight™ Bacterial Viability Kit, for microscopy and quantitative assays||LIVE/DEAD® Yeast Viability Kit|
|Target||Bacterial cell viability||Yeast cell viability|
|Reporter||Propidium iodide/SYTO® 9||FUN® 1/Calcofluor White|
|Ex/Em (nm)||485/530, 630||485/530, 620|
|Sample||Bacterial suspension||Pure or mixed cultures, body fluids, or environmental samples|
|Usage||Quick and easy two-color assay based on membrane integrity||Quick and easy two-color assay based on membrane integrity|
|Components||Bulk dye solutions||Bulk dye solutions|
|Format||1 kit, 10 microplates||1 kit, 10 microplates|
|Learn more from the Molecular Probes® Handbook