Microplate Assays for Cell Viability

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. 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. Assays involving MTT or XTT use that reducing power to generate a strongly colored reporter, formazan. 

We’ve developed alamarBlue and PrestoBlue assays to be simple and convenient by providing the reagents in a proprietary stabilizing formulation with a “add, incubate, and read” protocol that is scalable from single wells to high-throughput screening (HTS).

Our colorimetric viability assays, the Vybrant MTT assay and CyQUANT XTT assay, include all the reagents necessary to generate accurate and consistent colorimetric viability results.

 

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 or damaged plasma membranes allow for the influx of cell-impermeant dyes and the efflux or release of cytosolic enzymes into the surrounding media. Cell-impermeant DNA-binding dyes that fluoresce only when bound to DNA can be used as a cytotoxicity indicator that is independent of metabolic state. The release of soluble cytosolic enzymes, such as lactate dehydrogenase (LDH) or glucose 6-phosphate dehydrogenase release assay (G6PD) assays, into the supernatant can be easily quantified. We offer complete cytotoxicity kits for the detection of either LDH or G6PD release.

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.

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.

Selection guides
  alamarBlue Cell Viability Reagent PrestoBlue Cell Viability Reagent Vybrant MTT Cell Viability Assay CyQUANT XTT Cell Viability Assay
Application Detects metabolic activity; broad applicability and can be used with mammalian cells, bacteria, plant and fungi Primarily used to quickly detect metabolic activity within mammalian cells Detection of metabolic activity with mammalian cells Utilizes a soluble reaction product and an enhancer to detect metabolic activity within mammalian cells
Reporter Resazurin is converted to resorufin Resazurin is converted to resorufin Water-soluble MTT is converted into an insoluble purple formazan product Water-soluble XTT is converted into a water-soluble orange formazan product
Ex/Em (nm) 530–560/590 535–560/590–615 Absorbance detected at 570 nm Absorbance detected at 450 nm
Format 25 mL or 100 mL 25 mL or 100 mL 1 kit 1 kit
Protocol outline
  1. Add reagent to cells.
  2. Incubate at 37°C for 1–4 hours.
  3. Measure fluorescence.
  1. Add reagent to cells.
  2. Incubate at 37°C for 10 minutes to 4 hours.
  3. Measure fluorescence.
  1. Add reagent to cells.
  2. Incubate at 37°C for 4 hours.
  3. Add SDS-HCl soution; incubate at 37°C for 4 hours.
  4. Mix sample and read absorbance.
  1. Thaw XTT an dPMS reagents, mix.
  2. Add to cells; incubate at 37°C for 2–4 hours.
  3. Read absorbance.
Cat. No. DAL1025
DAL1100
A13261
A13262
V13154 X12223
  LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells Vybrant Cytotoxicity Assay Kit (G6PD release Assay) Pierce LDH Cytotoxicity Assay 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 INT tetrazolium salt is reduced to a red formazan product Image-IT DEAD Green, NuclearMask Deep Red or Hoechst 33342
Ex/Em (nm) 488/535 & 528/617 540 / 590 Absorbance detected at 490 nm and 680 nm 488/515, 638/686, or 350/461
Sample Adherent or suspended cells Cell suspension and surrounding media Cell suspension and surrounding medium 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 Reliable assay for the release of LDH from damaged cells Two-color assay based on plasma membrane integrity
Components Bulk dye solutions Complete assay Complete assay, including LDH control Complete assay
Format 1 kit, 10 microplates 1 kit, 10 microplates 200 or 1,000 reactions 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. Treat cells.
  2. Transfer supernatant to a new plate.
  3. Add reaction mixture.
  4. Incubate at room temperature for 30 min.
  5. Add stop solution.
  6. Measure absorbance at 490 nm and 680 nm.
  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 88953
88954
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
  alamarBlue Cell Viability Reagent PrestoBlue Cell Viability Reagent Vybrant MTT Cell Viability Assay CyQUANT XTT Cell Viability Assay
Application Detects metabolic activity; broad applicability and can be used with mammalian cells, bacteria, plant and fungi Primarily used to quickly detect metabolic activity within mammalian cells Detection of metabolic activity with mammalian cells Utilizes a soluble reaction product and an enhancer to detect metabolic activity within mammalian cells
Reporter Resazurin is converted to resorufin Resazurin is converted to resorufin Water-soluble MTT is converted into an insoluble purple formazan product Water-soluble XTT is converted into a water-soluble orange formazan product
Ex/Em (nm) 530–560/590 535–560/590–615 Absorbance detected at 570 nm Absorbance detected at 450 nm
Format 25 mL or 100 mL 25 mL or 100 mL 1 kit 1 kit
Protocol outline
  1. Add reagent to cells.
  2. Incubate at 37°C for 1–4 hours.
  3. Measure fluorescence.
  1. Add reagent to cells.
  2. Incubate at 37°C for 10 minutes to 4 hours.
  3. Measure fluorescence.
  1. Add reagent to cells.
  2. Incubate at 37°C for 4 hours.
  3. Add SDS-HCl soution; incubate at 37°C for 4 hours.
  4. Mix sample and read absorbance.
  1. Thaw XTT an dPMS reagents, mix.
  2. Add to cells; incubate at 37°C for 2–4 hours.
  3. Read absorbance.
Cat. No. DAL1025
DAL1100
A13261
A13262
V13154 X12223
  LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells Vybrant Cytotoxicity Assay Kit (G6PD release Assay) Pierce LDH Cytotoxicity Assay 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 INT tetrazolium salt is reduced to a red formazan product Image-IT DEAD Green, NuclearMask Deep Red or Hoechst 33342
Ex/Em (nm) 488/535 & 528/617 540 / 590 Absorbance detected at 490 nm and 680 nm 488/515, 638/686, or 350/461
Sample Adherent or suspended cells Cell suspension and surrounding media Cell suspension and surrounding medium 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 Reliable assay for the release of LDH from damaged cells Two-color assay based on plasma membrane integrity
Components Bulk dye solutions Complete assay Complete assay, including LDH control Complete assay
Format 1 kit, 10 microplates 1 kit, 10 microplates 200 or 1,000 reactions 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. Treat cells.
  2. Transfer supernatant to a new plate.
  3. Add reaction mixture.
  4. Incubate at room temperature for 30 min.
  5. Add stop solution.
  6. Measure absorbance at 490 nm and 680 nm.
  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 88953
88954
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

Resources

Learn more from the Molecular Probes Handbook

BioProbes Journal articles

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