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.

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.

CellEvent™ and PrestoBlue® reagents
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.

Selection guides
  alamarBlue® PrestoBlue® Cell Viability Reagent
Target Metabolic activity Metabolic activity
Reporter alamarBlue® (resazurin) Resazurin
Ex/Em (nm) 571/585 535–560/590–615
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
Protocol outline
  1. Add reagent to cells.
  2. Incubate at 37°C from 1–4 hours.
  3. Measure fluorescence.
  4. Analyze data.
  1. Add reagent to cells.
  2. Incubate at 37°C for 10 minutes.
  3. Measure fluorescence.
  4. Analyze data.
Cat. No. DAL1025 A13261
  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
Protocol outline
  1. Grow cells to desired density.
  2. Treat cells with test compound, incubate.
  3. Add staining reagents, incubate.
  4. Generate standard curve.
  5. Measure fluorescence at two wavelengths.
  6. Determine viability using standard curve.
  1. Grow cells to desired density.
  2. Treat cells with test compound, incubate.
  3. Add staining reagents, incubate.
  4. Generate standard curve.
  5. Measure fluorescence.
  6. Determine viability using standard curve.
  1. Plate cells.
  2. Add test compound or drug treatment.
  3. Add stain to cells.
  4. Incubate at 37°C for 30 min.
  5. Fix and permeabilize cells.
  6. Wash.
  7. Acquire images and analyze.
  1. Plate cells.
  2. Add test compound or drug treatment.
  3. Add stain to cells.
  4. Incubate at 37°C for 30 min.
  5. Fix and permeabilize cells.
  6. Wash.
  7. Acquire images and analyze.
Cat. No. L3224 V23111 I10291 H10290
  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
Protocol outline
  1. Grow bacteria to late log phase, pellet, resuspend in appropriate buffer.
  2. Divide resuspended bacteria and treat one sample with 70% isopropyl to kill bacteria.
  3. Mix live and killed bacteria to generate a standard curve for fluorescence ratio.
  4. Add bacteria to microplate wells at desired density.
  5. Apply experimental treatment to bacteria in microplate wells and incubate.
  6. Add staining reagents and incubate.
  7. Measure fluorescence.
  8. Determine percentage of live bacteria from standard curve.
  1. Treat yeast culture.
  2. Load yeast into microplate wells.
  3. Add FUN® 1 and Calcofluor White and incubate.
  4. Read fluorescence at both emission wavelengths.
  5. Calculate fluorescence ratio to determine metabolic activity.
Cat. No. L7012 L7009
For Research Use Only. Not for use in diagnostic procedures.