Larger dynamic range observed with the PrestoBlue HS compared to the standard PrestoBlue formulation

PrestoBlue HS and PrestoBlue Cell Viability Reagents are ready-to-use, non-toxic, resazurin-based solutions that function as cell health indicators to quantitatively measure viability. PrestoBlue uses the reducing power of living cells to convert resazurin to fluorescent resorufin. PrestoBlue HS (High Sensitivity) Cell Viability Reagent, a superior version of PrestoBlue, retains all the key characteristics that make PrestoBlue a great cell viability product, but now with improved sensitivity.

PrestoBlue HS and PrestoBlue are recommended in cases of quick viability determination since only a 10-minute incubation is required.

Selection guide

 PrestoBlue HS (high sensitivity) Cell Viability ReagentPrestoBlue Cell Viability Reagent
Use
  • Recommended for quick viability determination, only 10 minute incubation
  • Applicable to wide variety of mammalian cells
Mechanism of detection
  • Resazurin is converted to fluorescent resorufin
Sensitivity
Reagent purity
  • Purified
  • Varying concentrations of resorufin contamination depending on sources of material and manufacturing conditions
Background fluorescence
  • Low
  • >50% reduction in background fluorescence
  • Moderate
  • Increased background fluorescence due to presence of resorufin contamination
Signal-to-background
  • High
  • >100% ratio increase
  • Moderate
  • Reduced signal-to-background due to presence of resorufin contamination
Assay signal window
  • High
  • 2-fold increase in assay signal window compared to the non-HS assay
  • Moderate
  • Reduced dynamic range due to the presence of resorufin contamination
Cat. No.P50200A13262

PrestoBlue HS reagent is recommended for quick viability determination (10-minute incubation), whereas alamarBlue HS reagent is recommended in cases of extended viability studies or when using a high cell density.

Learn more about alamarBlue and alamarBlue HS

What are PrestoBlue assays?

Ideal for use on a variety cell types—including human, bacteria, and yeast cells—PrestoBlue can be used as an alternative viability detector in cell biology and microbiology (1). PrestoBlue is a quick cell viability indicator that uses the reducing power of live cells to convert resazurin to the fluorescent molecule, resorufin. Analysis can be evaluated quantitatively on an absorbance- or fluorescence-based microplate reader; while qualitative analysis can be evaluated by the visual color change of the solution which is indicative of metabolically active cells.

Resazurin is a non-toxic (Figure 1), cell permeable compound that is blue in color and virtually non-fluorescent. Within cells, resazurin is reduced to resorufin, which produces a highly fluorescent red color (Figure 2). Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability—and cytotoxicity.

Unlike other resazurin-based reagents, the PrestoBlue HS and PrestoBlue cell viability reagents have been formulated with a proprietary buffering system that results in a reagent with a physiological pH range optimal for the rapid determination of cellular viability.

Data shows same fluorescence viability signal with/without PrestoBlue HS

Figure 1. Demonstrated non-toxicity of PrestoBlue HS reagent. U2OS cells were treated with and without PrestoBlue HS reagent for 4 hours. CyQUANT Direct reagent was added to both populations following manufacturer’s instructions. Based on the CyQUANT Direct fluorescence measurements, there were no significant differences in fluorescence measurements, suggesting PrestoBlue HS reagent does not significantly contribute to cellular toxicity. Additionally, when exposed to varying concentrations of gambogic acid, the U2OS cells pre-treated with PrestoBlue HS reagent displayed similar IC50 values as the control cells.
 

Chemical structure conversion of resazurin and resorufin

Figure 2. Metabolically active cells convert resazurin to resorufin, a red-fluorescent indicator. Resazurin is a non-fluorescent indicator dye that undergoes chemical reduction to bright red-fluorescent resorufin in metabolically active cells. The amount of fluorescence produced is proportional to the number of living cells. In PrestoBlue HS, the resazurin is purified and provides superior performance compared to the standard PrestoBlue reagent which has known contamination issues that lead to sub-optimal performance.

How do I use PrestoBlue assays?

PrestoBlue HS and PrestoBlue cell viability reagent are quick and simple add-and-read assays (Figure 3). Add the PrestoBlue HS or PrestoBlue reagent to your cells, incubate for ≥10 minutes, and read the fluorescence or absorbance. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cell’s metabolic activity. Damaged and non-viable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells.

PrestoBlue HS and PrestoBlue cell viability assays are compatible with absorbance- and fluorescence-based microplate readers. Absorbance can be quantified at 570 nm and fluorescence can be quantified at 560/590 nm (excitation/emission). Results can be analyzed by plotting fluorescence intensity (or absorbance) versus compound concentration. While results are linear and quantitative for both fluorescence and absorbance, fluorescence readings provide higher sensitivity.

PrestoBlue workflow—reagent, microplate, incubation time, microplate reader, data graph
Figure 3. How PrestoBlue assays work.

PrestoBlue HS and PrestoBlue cell viability assays do not require cell lysis

PrestoBlue HS and PrestoBlue easily enter live cells, eliminating the need to lyse or further process cells using fixation and DNA denaturation techniques. The dye is stable in cell culture media, including media containing phenol red. These characteristics allow you to:

  • Continuously monitor the effects of drugs in dose response assay or when optimizing drug concentrations
  • Perform other functional assays after cell viability measurements with PrestoBlue HS or PrestoBlue are complete

 

PrestoBlue HS has improved performance

As a result of the synthesis and manufacturing processes, all resazurin-based reagents contain a detectable amount of highly fluorescent resorufin contamination. The amount of the resorufin contamination can vary greatly between sources of the material and manufacturing conditions. The varying amounts of resorufin contribute to differences in detectable background fluorescence. Additionally, the contaminating resorufin and the resulting higher background signal significantly reduces the signal-to-background ratio and dynamic range of the assay.

To improve the performance of the resazurin-based reagents, an innovative process was developed, and on implementation, removes the contaminating resorufin resulting in purified resazurin. To create PrestoBlue HS Cell Viability Reagent, the purified resazurin has been formulated with a proprietary buffering system.

PrestoBlue HS has higher sensitivity compared to PrestoBlue

The purified resazurin used for PrestoBlue HS reagent results in a >50% decrease in background fluorescence and a >100% increase in the signal-to-background ratio (Figures 4 and 5), while retaining all the key characteristics of PrestoBlue.

Figure 4. Lower background fluorescence in multiple cell types. The purified resazurin used for the PrestoBlue HS formulation results in a cell viability reagent displaying a background fluorescence reduction in adherent (U2OS), primary (HCASMC), and suspension (Ramos) cells.

Bar graph with 3 cell types showing PrestoBlue HS signal-to-background is ~3x higher than PrestoBlue

Figure 5. Performance improvement with various cell types. Improved cell viability signal-to-background ratios were displayed with HCASMC (primary cells), Ramos (suspension cells), and U2OS (adherent cells) when using the PrestoBlue HS cell viability reagent.

PrestoBlue HS has a larger assay window compared to PrestoBlue

The PrestoBlue HS reagent displays a larger viability detection window for various cell lines allowing for improved quantitative measurement of cell viability (Figure 6).

PrestoBlue HS signal-to-background is 2x greater than PrestoBlue, both decrease to 0 with increasing drug treatment
Figure 6. Increased dynamic range with PrestoBlue HS reagent. Various concentrations of gambogic acid were added to A549 cells and the changes in viability were detected. The results demonstrate a significant improvement in the dynamic range of the assay when using PrestoBlue HS reagent.
Thermo Scientific plate readers

Expertly detect fluorescence with Thermo Scientific plate readers

High-sensitivity fluorescence detection for 96–1,536 samples can be quickly performed on Fluoroskan or Fluoroskan FL Microplate Fluorometer or Varioskan LUX Multimode Microplate Reader using Invitrogen reagents for optimal detection. Take advantage of automatic dynamic range selection to get optimal gain settings for each individual well and automation capabilities for even higher throughput.

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Citations

Resources

Cell Analysis Learning Center—Find educational resources such as application notes, webinars, videos, articles, and more that cover the use of many of our reagents and kits for cell analysis.

Fluorescence SpectraViewer—Online tool for visualization of the excitation and emission of fluorescent reagents; allows for checking spectral compatibility for multiple fluorophores.

Application notes

 Application note: Evaluation of the toxic effects of PFOS in hESCs differentiating into cardiomyocytes

 Application note: Monitoring cell health with alamarBlue and PrestoBlue reagents using the Varioskan LUX Multimode Microplate Reader

BioProbes article

An Absorbance-Based Assay for Cell Health and Proliferation

Poster

 New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays

Support

Cell Analysis Support Center—Find technical information, tips and tricks, and answers to everyday problems.