alamarBlue Cell Viability Assay Protocol

Introduction

Cell health can be monitored by numerous methods. Plasma membrane integrity, DNA synthesis, DNA content, enzyme activity, presence of ATP, and cellular reducing conditions are known indicators of cell viability and cell death. The alamarBlue Cell Viability reagents, alamarBlue HS (high sensitivity) and alamarBlue, function as a cell health indicator by using the reducing power of living cells to quantitatively measure the proliferation of various human and animal cell lines, bacteria, plant, and fungi, allowing you to establish relative cytotoxicity of agents within various chemical classes. When cells are alive, they maintain a reducing environment within the cytosol of the cell. Resazurin, the active ingredient of the alamarBlue HS and alamarBlue reagents, is a non-toxic, cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Viable cells continuously convert resazurin to resorufin, increasing the overall fluorescence and color of the media surrounding cells.

As the result of the manufacturing process, all resazurin-based reagents contain a detectable amount of the highly fluorescent resorufin contamination. The amount of the contaminating resorufin can vary greatly in resazurin-based reagents and results in a high and variable background fluorescence reading. This higher background signal significantly reduces the signal-to-background ratio and the dynamic range of the assay.

To improve the performance of the resazurin-based reagents, an innovative process was developed to remove the contaminating resorufin, resulting in highly purify resazurin. This highly purified resazurin was used in the standard formulation creating the multiple component alamarBlue HS Cell Viability Reagent. Upon comparison to alamarBlue Cell Viability Reagent, the alamarBlue HS Cell Viability Reagent displays a >50% reduction in background fluorescence (Figure 1), a >100% increase (Figure 2) in the signal-to-background ratio and a large increase in the dynamic range (Figure 3).

alamarBlue HS reagent background reduction as shown in bar chart

Figure 1. Reduction of background fluorescence displayed with alamarBlue HS reagent compared to alamarBlue reagent. A 100 µL aliquot of complete growth media was added to several wells of a 96 well plate. A 10 µL aliquot of either the alamarBlue HS or alamarBlue reagent was added to the wells containing complete growth media and the fluorescence was detected at various times after reagent was added. The alamarBlue HS consistently displays a >50% reduction in background fluorescence.

alamarBlue HS reagent signal-to-background as shown in bar chart

Figure 2. alamarBlue HS displays a significant increase in the signal-to-background ratio. A549 cells were seeded at 5,000 cell/well and incubated overnight to allow for attachment. A 10 µL aliquot of alamarBlue HS or alamarBlue reagent was added to the wells containing 100 µL of complete growth media and the attached cells. At various time points after reagent addition the fluorescence was measured. The results demonstrate the >100% increase in the signal-to-background ratio for the alamarBlue HS versus the alamarBlue reagent.

alamarBlue HS reagent signal linearity as shown in graph

Figure 3. Large increase in dynamic range displayed with the alamarBlue HS Cell Viability Reagent. A549 cells were seeded at 5,000 cells/well and incubated overnight to allow for attachment. Various concentrations of gambogic acid were added to A549 cells. After incubation, 10 µL of either alamarBlue or alamarBlue HS reagent was added to the wells and the fluorescence was measured and graphed as signal to background. The results demonstrate a significant improvement in the dynamic range of the assay when using alamarBlue HS reagent.

The alamarBlue Cell Viability Reagent is used to assess cell viability by simply adding the 10X, ready-to-use solution to mammalian or bacterial cells in culture media. Since the formulation is consistent for both alamarBlue and alamarBlue HS, their assay protocols are therefore the same. There is no requirement to aspirate media from cells or place cells in minimal media. Consequently, the alamarBlue reagent can easily be used in a single tube or microtiter plate format in a “no wash” fashion. Simply add either alamarBlue reagent as 10% of the sample volume (i.e., add 10 μL alamarBlue reagent to 100 μL sample), followed by a 1–4 hour incubation at 37°C. Longer incubation times may be used for greater sensitivity without compromising cell health (see Frequently Asked Questions). The resulting fluorescence is read on a plate reader or fluorescence spectrophotometer. Alternatively, the absorbance of alamarBlue reagents can be read on a spectrophotometer. Finally, results are analyzed by plotting fluorescence intensity (or absorbance) versus compound concentration (Figure 4).

alamarBlue HS and alamarBlue Cell Viability Reagents assay protocol

Figure 4. alamarBlue HS and alamarBlue Cell Viability Reagents assay protocol. A 96-well plate containing the cells and the compounds to be tested is prepared using standard methods. Either alamarBlue reagent is added directly to each well, the plates are incubated at 37°C to allow cells to convert resazurin to resorufin, and the fluorescence (or absorbance) signal is measured. Results are evaluated by subtracting the background fluorescence from the signal. The signal to background ratio is then plotting versus compound concentration.

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Materials

Materials required but not provided

  • Mammalian or bacterial cells in appropriate medium
  • 96- or 384-well plates
  • Optional: 3% SDS in phosphate buffered saline (PBS), pH 7.4

Preparing cells

  • Mammalian cells—adherent: One day before your experiment, seed cells into a 96-well plate containing 100 µL/well of cell culture medium.
    • Note: If you are using a 384-well plate, use 50 µL/well of cell culture medium.
  • Mammalian cells—suspension: seed cells into a 96-well plate containing 100 µL/well of cell culture medium. The viability of the cells can be determined immediately or grow for up to 24 hours at 37°C and 5% CO2 before proceeding with the assay.
  • Bacterial cells: For details, see references 2 and 3.

General guidelines

  • Both alamarBlue HS and alamarBlue Cell Viability reagents are able to detect changes in viability from a wide variety of organisms, including human and animal cell lines, bacteria, plant, and fungi
  • Be sure to include appropriate assay controls. To minimize experimental errors, we recommend making measurements from a minimum of 4–8 replicates of experimental and no-cell control samples.
  • You may need to determine the optimal plating density and incubation time for the alamarBlue and alamarBlue HS assays for each cell type and use conditions such that the assay is in the linear range.
  • If you plan to use longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to avoid microbial contaminants. Contaminated cultures will yield erroneous results as microbial contaminants also reduce alamarBlue reagents.
  • Fetal bovine serum (FBS) and bovine serum albumin (BSA) may cause minor quenching of fluorescence. We recommend using the same serum concentration in the no cell controls to account for this quenching. Other media components, such as phenol red do not interfere with the assay.

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Protocol

Optional: Treat cells with the test compound 24–72 hours prior to performing the alamarBlue HS or alamarBlue cytotoxicity assay.

  1. Add 1/10th volume of either alamarBlue HS or alamarBlue reagent directly to cells in culture medium as described in the table below.
Format
Volume of cells + medium
Volume of 10x alamarBlue HS or alamarBlue reagent to add
Cuvette
1 mL
100 µL
96-well plate
100 µL
10 µL
384-well plate
40 µL
4 µL
  1. Incubate for 1 to 4 hours at 37°C in a cell culture incubator, protected from direct light.
    • Note: Sensitivity of detection increases with longer incubation times. For samples with fewer cells, use longer incubation times of up to 24 hours.
  2. Record results using fluorescence or absorbance as follows:
    • Fluorescence: Read fluorescence using a fluorescence excitation wavelength of 540–570 nm (peak excitation is 560 nm). Read fluorescence emission at 580–610 nm (peak emission is 590 nm).
    • Absorbance: Monitor the absorbance of alamarBlue at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).
    • Note: Fluorescence mode measurements are more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarBlue reagent. Assay plates or tubes can be wrapped in foil, stored at 4°C, and read within 1–3 days without affecting the fluorescence or absorbance values.
  3. Optional: Add 50 μL 3% SDS directly to 100 μL of cells in alamarBlue reagent to stop the reaction.
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Frequently asked questions (FAQs)

General questions

Q: How does alamarBlue work?
A: Healthy living cells maintain a reducing state within their cytosol. This “reducing potential” of cells converts alamarBlue reagent into a detectable fluorescent (or absorbent) product.

Q: Is alamarBlue reagent toxic?
A: No. alamarBlue reagent is a safe, non-toxic reagent to both the sample and user. After viability testing with alamarBlue replace the media with fresh growth media and incubate. Cells we proliferation and can been split and seeded on their normal schedule.

Q: Does alamarBlue reagent need reconstitution?
A: No, alamarBlue reagent is supplied as a 10X, ready-to-use solution.

Q: Can I use alamarBlue reagent with suspension cells too?
A: Yes. alamarBlue reagent works on adherent and suspension mammalian cells.

Q: Can I use alamarBlue reagent with non-mammalian cells, such as bacteria?
A: Yes, alamarBlue reagent has been shown to work with bacterial2 and plant cells4.

Storage questions

Q: What if I left the alamarBlue reagent at room temperature overnight?
A: The reagent is stable for up to 12 months when stored at room temperature (~22ºC).

Q: I accidentally froze the alamarBlue stock reagent, can I still use it?
A: Yes. alamarBlue HS and alamarBlue reagents are stable to multiple freeze/thaw cycles. Be sure to heat the reagent in a 37°C water bath and mix the reagent to ensure a homogenous solution before use.

Q: Do I need to protect alamarBlue reagent from light?
A: Yes, alamarBlue reagents are very slowly converted into a fluorescent product over time, when exposed to light, thus leading to high background values. Store the reagent, protected from light.

Methods questions

Q: What is the optimal incubation time and temperature of cells with alamarBlue reagent?
A: Incubate the cells with alamarBlue reagent for 1–4 hours at 37°C. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours.

Q: Can you incubate cells with alamarBlue reagent overnight?
A: Yes. However, signals from higher cell density samples may have "saturated," which means the linearity of reagent may have reached a plateau. If this occurs, decrease the incubation time.

Q: What if I don’t have an instrument suitable for reading fluorescence?
A: The absorbance of alamarBlue reagent also changes depending on cell viability and proliferation. Therefore, simply monitor the absorbance of the reagent at 570 nm, while using 600 nm as a reference wavelength.

Q: Is alamarBlue assay strictly an endpoint assay?
A: No. While alamarBlue can be used as a terminal readout of a population of cells, the reagent can also be used to continuously monitor cell viability and proliferation in real time. Since alamarBlue reagent is non-toxic, you can incubate cells with reagent and monitor fluorescence (or absorbance) over time on the same sample.

Troubleshooting questions

Q: What is the problem for observing high background fluorescence values?
A: The reagent may be breaking down due to exposure to light. Be sure to store alamarBlue reagent in the dark and do not expose the reagent to direct light for long periods of time.

Q: Why are the fluorescence values so low in intensity?
A: Try increasing the incubation time of cells with alamarBlue reagent, changing the instrument’s "gain" setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (living cells) in the experimental design for troubleshooting.

Q: Why are the fluorescence values so high that they are beyond the linear range of the instrument?
A: Try decreasing the incubation time or reducing the number of cells used in the experiment.


References