alamarBlue Assays for Cell Viability

Highly referenced for cytotoxicity and viability assays, alamarBlue cell viability assay has been used for years in biological and environmental studies (1). Furthermore, analysis of cell proliferation and cytotoxicity is a vital step in evaluating cellular health and in the drug discovery process. alamarBlue is a proven cell viability indicator that uses the natural 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.

The active ingredient of alamarBlue (resazurin) is a non-toxic (Figure 1), cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces a very bright red fluorescence (Figure 2). Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability—and cytotoxicity.

Figure 1. Demonstrated Non-Toxicity of the alamarBlue HS Cell Viability Reagent. U2OS Cells were treated with and without alamarBlue HS 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 alamarBlue HS does not significantly contribute to cellular toxicity. Additionally, when exposed to varying concentrations of gambogic acid, the U2OS cells pre-treated with alamarBlue HS displayed similar IC50 values as the control cells.

alamarBlue has a proven track record as an indicator of cell health and its nontoxic nature permits long-term exposure of cells without negative impact; cells grown in the presence of alamarBlue were found to produce similar numbers of viable cells as control cells, as determined by flow cytometric analysis of CD44, CD45RB, and CD4 antigens.

The alamarBlue HS reagent refers to the fact that the alamarBlue HS reagent contains purified resazurin, devoid of any contaminating resorufin which can negatively impact reagent performance. The alamarBlue HS reagent provides a >50% reduction in background signal and >100% signal-to-background ratio increase compared to the standard alamarBlue reagent, while retaining all the key characteristics that make alamarBlue a highly-referenced cell viability and cytotoxicity reagent.

TOP

alamarBlue HS and alamarBlue cell viability reagents are easy add-and-read assays (Figure 3). Simply add alamarBlue HS or alamarBlue to your cells, incubate for 1–4 hours, 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.

alamarBlue assays are compatible with multiple instrument platforms. After incubation with alamarBlue HS or alamarBlue, your samples can readily be measured on fluorescence and absorbance instrumentation. For fluorescence, simply set up your plate reader or fluorescence spectrophotometer using 560/590 nm (excitation/emission) filter settings. Alternatively, the absorbance of alamarBlue HS and alamarBlue can be read on a UV-Vis spectrophotometer at 570 nm.

Finally, analyze results 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 alamarBlue assays work.

alamarBlue HS easily enters into 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 a dose response assay or when optimizing drug concentrations
• Perform other functional assays after cell viability measurements with alamarBlue HS or alamarBlue are complete

TOP

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 help improve the performance of the resazurin-based reagents, an innovative process was developed and, on implementation, removes the contaminating resorufin resulting in purified resazurin. This purified resazurin was used in the standard formulation creating the multiple component alamarBlue HS Cell Viability Reagent.

The purified resazurin used for alamarBlue HS reagent results in a >50% decrease in background fluorescence and a >100% increase in the signal-to-background ratio (Figures 4 and 5).

Figure 4. Significantly lower background fluorescence observed when comparing alamarBlue HS vs alamarBlue. The purified resazurin used for the alamarBlue HS formulation results in a cell viability reagent displaying a background fluorescence reduction in suspension (Ramos), primary cells (HCASMC), and adherent (U2OS) cells.

The alamarBlue HS reagent displays an extended viability detection time window for a wide variety of organisms and thus can be used with various human and animal cell lines, bacteria, plant, and fungi, resulting in quantitative measurement of cell viability and proliferation (Figure 6).

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

TOP

1. Rampersad SN (2012). Sensors. 12(9):12347-12360. PMID: 23112716