Invitrogen™ PrestoBlue™ and PrestoBlue™ HS Cell Viability Reagents

This depends upon the matrix material. To determine if the matrix material binds the dye non-specifically, we recommend incubating the matrix material alone (no cells) with the reagent, decanting and seeing if there is excessive staining of the matrix material.

One reason could be that the dye in your PrestoBlue reagent has precipitated resulting in varying dye concentration. In such cases, PrestoBlue reagent should be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution. Another cause can be pipetting issues. Ensure that your pipettor has been calibrated and the pipette tips are securely anchored prior to pipetting.

Decrease the incubation time or reduce the number of cells used in the experiment.

We recommend increasing the incubation time of cells with PrestoBlue reagent, changing the instrument's gain or voltage setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (living cells) in the experimental design for troubleshooting.

The reagent may be breaking down due to exposure to light. Be sure to store PrestoBlue reagent in the dark and do not expose the reagent to direct light for long periods of time.

Because the indicator is a multicomponent solution, we recommend that frozen PrestoBlue reagent be warmed to 37 degrees C and shaken or swirled to ensure that all components are completely in solution.

The reagent is stable for up to 12 months when stored at room temperature (approximately 22 degrees C).

Yes. PrestoBlue reagent is stable to multiple freeze/thaw cycles. Be sure to heat the reagent in a 37 degrees C water bath and mix the reagent to ensure a homogenous solution before use.

It is possible to combine PrestoBlue reagent (for cell proliferation) with the CellEvent Caspase 3/7 Green Detection Reagent (for apoptosis detection) in the same sample.

alamarBlue reagent and PrestoBlue reagent contain resazurin in a proprietary stabilizing formulation that allows for a convenient “mix, incubate, and read” protocol. PrestoBlue reagent is an improvement in the formulation of alamarBlue reagent that allows for much faster staining (typically 10 minutes vs. 1-4 hours to obtain a similar signal and sensitivity). C12-resazurin is a derivative of resazurin that has better cellular retention and thus allows for analysis on a flow cytometer and multiplexing with viability indicators and other biomarkers.

alamarBlue Cell Viability Reagent, PrestoBlue Cell Viability Reagent, and CyQUANT Cell Proliferation Assay Kit (Cat. No. C7026) have been validated for use with the AlgiMatrix 3D Culture System and should also work with other 3D culture systems. For further details, please refer to this poster (https://aacrjournals.org/cancerres/article/70/8_Supplement/3234/563757/Abstract-3234-AlgiMatrixT-3-D-cell-culture-system) and protocol (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/3-d-cell-culture-protocol/algimatrix-viability-using-cyquant.html).

No, it is not possible to assay the viability of the same population of cells longer than a few hours; you will need to use replicate samples. DNA binding dyes are toxic to cells; stained cells should be imaged as soon as possible after staining. Calcein AM is not retained in cells and may be actively effluxed out in the range from minutes to several hours, dependent upon the cell type. Calcein AM is not toxic to cells, so it can be added repeatedly to the same samples. You can assay the proliferation of the same sample of cells over several days using alamarBlue reagent or PrestoBlue reagent, as these dyes are non-toxic to cells.

Fetal bovine serum (FBS) and bovine serum albumin (BSA) cause some quenching of fluorescence. We recommend using the same serum concentration in controls to account for this quenching. Assuming the media does not contain reducing agents, other media components do not interfere with the assay. There is no interference from the presence of phenol red in the growth medium if the media pH has not changed significantly. At or near neutral pH, the presence of phenol red merely shifts the values approximately 0.03 units higher. Ideally, the assay should be done in phenol red-free medium.

Fluorescence: Read fluorescence using an excitation wavelength between 530-570 nm (peak excitation is 570 nm). Read fluorescence emission between 580-610 nm (peak emission is 585 nm).
Absorbance: Monitor the absorbance of PrestoBlue reagent at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).
Note: Fluorescence detection is more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of PrestoBlue reagent. With most animal cells, assay plates or tubes can be wrapped in foil or plastic wrap (to prevent evaporation), stored at 4 degrees C, and read within 1-3 days without affecting the fluorescence or absorbance values. This may not hold true for bacterial, algal, protozoan, or fungal samples.

You may need to determine the plating density and incubation time for the PrestoBlue assay for each cell type and use conditions such that the assay is in the linear range.

Plating Density:
PrestoBlue reagent measures cell proliferation most accurately when the cells are in the exponential growth phase. If the cell density is too high, cell proliferation will decrease, giving less reduction of the PrestoBlue regent than would have been expected. At low cell density, the slower growth rate could result in insignificant PrestoBlue reduction. A cell density of 1 x 10E4 cells/mL is generally recommended for animal cells, with cells in the exponential phase. However, since cells vary in their proliferation rate, it is impossible to recommend a cell density that is suitable for all experiments. Instead we recommend that you perform a control experiment to determine the optimum cell density for your studies.

Incubation Time:
It is equally important to optimize the incubation time for the PrestoBlue reagent. Incubate the cells with PrestoBlue reagent for 1-4 hours at 37 degrees C or, at an optimal temperature for the species/cell type. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours. If you plan to use a longer incubation time (overnight), be sure to maintain sterile conditions during reagent addition and incubation to minimize the introduction of microbial contaminants. Contaminated cultures will yield erroneous results, as microbial contaminants also reduce PrestoBlue reagent.

PrestoBlue reagent contains proprietary buffering agents that maintain the stability and solubility of both the oxidized and reduced forms of the PrestoBlue reagent and other components in the solution. Over extended incubation times with cells, the buffering capacity may be altered to such an extent that reagent solubility and stability may be affected, resulting in a loss of signal.
Alternatively, if the length of the experiment is longer than the optimal PrestoBlue incubation time, we suggest that an endpoint test is used. This type of experiment is particularly useful for cell proliferation studies over days, weeks, and months. PrestoBlue reagent can be used for long-term cell proliferation studies where measurements are taken repeatedly. For these types of studies, we recommend that aliquots of the cell medium/ suspension are taken at each time point during incubation with PrestoBlue reagent prior to an endpoint.
If your test compound has delayed cytotoxic effects, (i.e., cells will not show a response for several hours or even days), we advise adding the PrestoBlue reagent toward the end of the intended exposure period, after cells have been affected. If the PrestoBlue reagent is added at the start of your experiment, viable cells will reduce resazurin before your compound can take effect, thus compromising your end results.
If loading multiple wells of a 96- or 384-well plate using a single-channel pipette, add PrestoBlue reagent to all wells containing the same condition prior to moving to next condition for most consistent results. Begin timing after adding reagent to the last well on the plate.

We recommend making measurements from a minimum of 4-8 replicates of experimental and no-cell control samples. Some suggestions are provided below:

No-cell controls: Wells that contain only culture media so that the background fluorescence can be determined and subtracted from experimental wells. We recommend using the same serum concentration in controls. Phenol red may interfere with the assay; ideally the assay should be performed using phenol red-free media.
No-cell + experimental compound controls: Wells that contain only cell culture media and added compound to assess potential background fluorescence or absorbance caused by the drug/chemical used in the experiment. Untreated cell controls: If you're treating cells with a compound, we recommend that you plate wells of untreated cells.
Solvent controls: If you're treating cells with a compound dissolved in a solvent such a DMSO, ethanol, etc., we recommend that you plate wells of cells with added solvent only (same volume added to experimental samples, no compound); this control should provide similar results as untreated cell controls. If results from these samples are different from untreated cell controls, the sample may be sensitive to the solvent.

PrestoBlue reagent has been used with many different cell species: mammalian, avian, amphibian, fish, diatoms, bacteria, plant cells, fungi and others-as well as cell samples, such as immortalized cell cultures, cancer cells, stem cells, primary cell cultures, and suspension cell cultures. Also, PrestoBlue reagent has been used to measure viability of tissue samples such as heart valves and cells in 3D matrices such as AlgiMatrix matrix. There are many references available for this application. You can find them by searching for PrestoBlue and your cell type of interest. For example, using Google Scholar, use the search term “PrestoBlue and cancer cells” or PrestoBlue and tissue”.

PrestoBlue reagent can be either a live-cell or end-point assay. PrestoBlue reagent allows you to develop live-cell assays for real-time monitoring of cell metabolism and viability without using any hazardous solvents, or requiring disposal of scintillation cocktail and radioactive waste. Assayed cells can be recovered for further culturing or use in subsequent assays. Alternatively, for most animal cells, assay plates or tubes can be wrapped in foil, stored at 4 degrees C, and read within 1-3 days without affecting the fluorescence or absorbance values. This may not hold true for bacterial, algal, protozoan, or fungal samples.

If an end-point assay is preferred, it is possible to stop and stabilize the reaction by the adding 3% SDS (50 µL of 3% SDS for every 100 µL original culture volume is sufficient). The plate can then be stored at room temperature for up to 48 hours before recording data, provided that the contents are protected from light and sealed to prevent evaporation. Cells cannot be further cultured after this step.

You will not have any problems with PrestoBlue reagent left at room temperature overnight. PrestoBlue reagent is stable for at least 3 months at room temperature (approximately 22 degrees C) when stored protected from light.

You can still use PrestoBlue reagent after it has been frozen. PrestoBlue reagent has been tested after 5 freeze/thaw cycles with no change in assay performance. Be sure to thaw the reagent completely and mix it so that the solution is homogenous before use.

Both resazurin and resorufin are light sensitive. Storage in the brown bottle is recommended as prolonged exposure (greater than a month) of PrestoBlue reagent to light will increase the background fluorescence and decrease assay sensitivity. Background fluorescence can be corrected for by including no-cell control wells, but the assay window will be decreased.

No, PrestoBlue reagent does not need to be reconstituted. It comes as a 10x, ready-to-use solution that can be added directly to the cells in culture media. Simply add 10 µL of reagent to 90 µL of sample.

PrestoBlue Cell Viability Reagent is sensitive enough to detect 10 animal cells in a single well of a 384-well plate.

While most reports in the literature suggest that solutions containing resazurin, the active ingredient in PrestoBlue reagent, are not toxic to cells, other reports clearly show that cell viability is affected depending on the length of exposure and concentration of resazurin to which they are subjected. In our experience, it is unlikely that cells will be adversely affected by exposure to PrestoBlue reagent if assay conditions remain within the suggested incubation period (i.e., 10 min to 2 hr). Cells can be incubated up to 24 hr in the presence of PrestoBlue reagent, and then further cultured. Simply remove the reagent from cells and replace it with growth medium.

PrestoBlue Cell Viability Reagent is a proven cell viability and proliferation indicator. It contains the active ingredient, resazurin, a non-toxic, cell permeable, non-fluorescent blue indicator dye. Healthy living cells maintain a reducing state within their cytosol. Upon diffusion into cells, the natural reducing potential of living cells converts resazurin to the very bright red, cell permeable fluorescent dye, resorufin. Resazurin is reduced by the removal of oxygen.

Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability, proliferation - and cytotoxicity. The net result is measurable change in the color and fluorescence intensity. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cells' metabolic activity. Damaged and nonviable cells have lower metabolic activity and thus generate a proportionally lower signal than healthy cells.

Fetal bovine serum (FBS) and bovine serum albumin (BSA) cause some quenching of fluorescence. We recommend using the same serum concentration in controls to account for this quenching. Assuming the media does not contain reducing agents, other media components do not interfere with the assay. There is no interference from the presence of phenol red in the growth medium if the pH has not changed significantly. At or near neutral pH, the presence of phenol red merely shifts the values approximately 0.03 units higher. Ideally, the assay should be done in phenol red-free medium.

You may need to determine the plating density and incubation time for the alamarBlue assay for each cell type and use conditions such that the assay is in the linear range.

Plating Density:
alamarBlue reagent measures cell proliferation most accurately when the cells are in the exponential growth phase. If the cell density is too high, cell proliferation will decrease, giving less reduction of the alamarBlue reagent than would have been expected. At low cell density, the slower growth rate could result in insignificant alamarBlue reduction. A cell density of 1 x 10E4 cells/mL is generally recommended for animal cells, with cells in the exponential phase. However, since cells vary in their proliferation rate, it is impossible to recommend a cell density which is suitable for all experiments. Instead we recommend performing a control experiment to determine the optimum cell density for your studies. A detailed protocol is provided in the product manual.

Incubation Time:
It is equally important to optimize the incubation time for the alamarBlue reagent. Incubate the cells with alamarBlue reagent for 1-4 hours at 37 degrees C or, at an optimal temperature for the species/cell type. For more sensitive detection with low cell numbers, increase the incubation time for up to 24 hours. 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 reagent. A detailed protocol is provided in the product manual.

alamarBlue reagent contains proprietary buffering agents which maintain the stability and solubility of both the oxidized and reduced forms of the alamarBlue reagent and other components in the solution. Over extended incubation times with cells, the buffering capacity may be altered to such an extent that reagent solubility and stability may be affected, resulting in a loss of signal.

Alternatively, if the length of the experiment is longer than the optimal alamarBlue incubation time, we suggest that an endpoint test is used. This type of experiment is particularly useful for cell proliferation studies over days, weeks and months. alamarBlue reagent can be used for long-term cell proliferation studies where measurements are taken repeatedly. For these types of studies, we recommend that aliquots of the cell medium/ suspension are taken at each time point during incubation with alamarBlue reagent prior to an endpoint.

We recommend including 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. Some suggestions are provided below:

No-cell controls: Wells that contain only culture media so that the background fluorescence can be determined and subtracted from experimental wells. We recommend using the same serum concentration in controls. Phenol red may interfere with the assay; ideally the assay should be performed using Phenol red-free media.
No-cell + experimental compound controls: Wells that contain only cell culture media and added compound to assess potential background fluorescence or absorbance caused by the drug/chemical used in the experiment.
Untreated-cell controls: If you're treating cells with a compound, we recommend that you plate wells of untreated cells.
Solvent controls: If you're treating cells with a compound dissolved in a solvent such a DMSO, ethanol, etc., we recommend that you plate wells of cells with added solvent only (same volume added to experimental samples, no compound); this control should provide similar results as untreated cell controls. If results from these samples are different from untreated cell controls, the sample may be sensitive to the solvent.

Fluorescence: Read fluorescence using an excitation wavelength between 530-570 nm (peak excitation is 570 nm). Read fluorescence emission between 580-610 nm (peak emission is 585 nm).
Absorbance: Monitor the absorbance of alamarBlue reagent at 570 nm, using 600 nm as a reference wavelength (normalized to the 600 nm value).
Note: Fluorescence detection is more sensitive. When fluorescence instrumentation is unavailable, monitor the absorbance of alamarBlue reagent. With most animal cells, assay plates or tubes can be wrapped in foil or plastic wrap (to prevent evaporation), stored at 4 degrees C, and read within 1-3 days without affecting the fluorescence or absorbance values. This may not hold true for bacterial, algal, protozoan, or fungal samples.

alamarBlue Cell Viability Reagent compound is light sensitive and should be stored in the dark. The product may be stored for 12 months at room temperature. The expiration date is given on the product label. If shelf life beyond 12 months is desired, storing at 2-8 degrees C increases shelf life to 20 months. alamarBlue reagent may also be frozen at greater than -70 degrees C indefinitely. Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37 degrees C and shaken to ensure all components are completely in solution.

alamarBlue reagent has been used with many different cell species: mammalian, avian, amphibian, fish, diatoms, bacteria, plant cells, fungi and others-and cell samples, such as immortalized cell cultures, primary cell cultures, cancer cells, stem cells, etc. Also, alamarBlue reagent has been used to measure viability of tissue samples such as heart valves and cells cultured in 3D matrices such as AlgiMatrix matrix. There are many references available for this application. You can find them by searching for alamarBlue and your cell type of interest. For example, using Google Scholar, use the search term ‘alamarBlue and cancer cells' or ‘alamarBlue and tissue'.

While most reports in the literature suggest that solutions containing resazurin, the active ingredient in alamarBlue reagent, are not toxic to cells, other reports clearly show that cell viability is affected depending on the length of exposure and concentration of resazurin to which they are subjected.

alamarBlue Cell Viability Reagent is a proven cell viability and proliferation indicator. It contains the active ingredient, resazurin, a non-toxic, cell permeable, non-fluorescent blue indicator dye. Healthy living cells maintain a reducing state within their cytosol. Upon diffusion into cells, the natural reducing potential of living cells converts resazurin to the very bright red, cell permeable fluorescent dye, resorufin. Resazurin is reduced by the removal of oxygen.

Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability, proliferation, and cytotoxicity. The net result is measurable change in the color and fluorescence intensity. The amount of fluorescence or absorbance is proportional to the number of living cells and corresponds to the cells' metabolic activity. Damaged and nonviable cells have lower metabolic activity and thus generate a proportionally lower signal than healthy cells.

PrestoBlue Cell Viability Stain and CyQUANT Cell Proliferation Assay Kit can be used. We also offer a Neurite Outgrowth Staining Kit (Cat. No. A15001). More information about our different assays for neuronal cell health can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-analysis/neuroscience/neuronal-cell-health-assays.html).

Other than the Invitrogen alamarBlue Cell Viability Reagent, AlgiMatrix Matrix is also compatible with Invitrogen PrestoBlue Cell Viability Reagent and Invitrogen CyQuant Direct Cell Proliferation Assay. The protocol is almost the same as the 2D cell culture, but it needs the blank AlgiMatrix Matrix as a blank control.