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.
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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.
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.
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.
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 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.
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).
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.
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.
Cell Analysis Support Center—Find technical information, tips and tricks, and answers to everyday problems.
5 Steps resources
For Research Use Only. Not for use in diagnostic procedures.