PrestoBlue Assays for Cell Viability

Excellent 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.
 

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.

Figure 3. How PrestoBlue assays work.

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

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 to help reduce 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.

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.

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

Also see poster:  New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays

1. Lall N, Henley-Smith CJ, de Cana MN, et al (2013). Int J Microbiol. 2013:420601. PMID: 23653650