Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support—Getting Started

No, the tissue was not cryopreserved.

Please see our protocol here for thawing frozen cells

Synth-a-Freeze Cryopreservation Medium can be used with any standard freezing protocol. It offers performance comparable to that of our standard, serum-containing cryopreservation medium for cyropreserving a variety of cell types including human keratinocytes, embryonic stem cells, neural stem cells, and mesenchymal stem cells. 

There isn’t a Drug Master File, but there is a Device Master File on file with the FDA. Please contact our Licensing team at outlicensing@thermofisher.com in order to obtain instructions for referencing this Device Master File.

We recommend using DMEM plus 10% FBS and 10% DMSO, or the Recovery Cell Culture Freezing Medium. You can try the same general recommendations for freezing cells, just changing the freezing solution. 

There have not been any bench studies at this time. It is best to aliquot this product.

Recovery Cell Culture Freezing Mediumshould be stable at 2 to 8°C for 1 week, although we have no data to support this.

Recovery Cell Culture FreezingMedium is meant for general cell culture applications where a wide range of growth media will be used. We recommend that you aspirate Recovery Cell Culture Freezing Mediumbefore seeding, although other protocols can be substituted (this will need to be determined by the end user).

Trypan blue is a cell impermeant stain used to estimate the number of dead cells in a viable population. Its utility is based on the fact that it is a charged dye and does not enter cells unless the membrane is compromised. Live (viable) cells exclude the dye but dead (non-viable) cells or cells with a compromised membrane are stained an intense blue.

Trypan blue will bind to serum proteins as well as cellular proteins, which may result a high level of background staining. If the background is too dark, cells should be pelleted and resuspended in protein-free medium, buffer, or normal saline prior to counting.

We recommend adding 0.1 mL of trypan blue stock solution to 1 mL of cells (final concentration 0.04% w/v). Final concentrations may range from 0.0.04% to 0.2% (w/v) dependent upon the sample and instrumentation. 

Yes, you can find the protocol at the following link.

Trypan blue will stain cells that have a compromised membrane. It cannot differentiate between compromised membranes caused by apoptosis or necrosis.

It depends on the type of fluorescent stain used on the cells. Trypan blue is a cell-impermeant chromophore that can quench fluorescence. It may quench fluorescent staining on the surface of live cells or internal fluorescent staining in dead cells.

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.

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 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’.

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°C increases shelf life to 20 months. alamarBlue reagent may also be frozen at >–70°C indefinitely. Because the indicator is a multicomponent solution, we recommend that frozen alamarBlue reagent be warmed to 37°C and shaken to ensure all components are completely in solution.

• 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°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. 

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. 

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°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. 

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. 

Our alamarBlue™ reagent contains resazurin in a proprietary stabilizing formulation that allows for a convenient “mix, incubate, and read” protocol. C₁₂-resazurin is a derivative of resazurin that has better cellular retention due to the twelve-carbon alkyl chain and thus allows for analysis on a flow cytometer and multiplexing with viability indicators and other biomarkers.

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.

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 sensitive enough to detect 10 animal cells in a single well of a 384-well plate.

Serial two-fold dilutions of Jurkat cells from 0–100,000 were incubated withPrestoBlue™ reagent at 37°C/5% CO₂ for 10 minutes and 16 hours. For the 10-minute incubation, the signal from 98 cells was greater than the no cell control ±3 standard deviations (A). For the 16-hour incubation, the signal from12 cells was greater than the no cell control ±3 standard deviations (B).

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.

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.

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.

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 (~22°C) when stored protected from light.

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°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.

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”. 

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. 

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°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.

• 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°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. 

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. 

• The LIVE/DEAD™ Fixable kits for flow cytometry analysis are compatible with fixation. These kits use amine-reactive cell-impermeant dyes that stain the cell surface of live cells and also the cytosol of dead cells—live cells are dim and dead cells are bright. Since the dye is covalently bound to the cells, it will be retained after fixation. Unfortunately, this method does not work well for imaging-based assays, as all cells are stained and it is difficult to distinguish bright dead cells from dim live cells with a microscope.
• Image-IT™ DEAD Green Viability Stain (Cat. No. I10291) for imaging and high-content screening (HCS) analysis is a live-cell impermeant DNA binding dye that is compatible with fixation and permeabilization with good retention up to 48 hours. 
• We also have a LIVE/DEAD™ Reduced Biohazard Cell Viability Kit (Cat. No. L7013) for imaging and flow analysis that contains two DNA binding dyes, SYTO™ 10 and Dead Red, that are sufficiently retained to be analyzed soon after 4% glutaraldehyde fixation. 

Note: In general, DNA-binding dyes and calcein AM are not compatible with fixation, as these dyes are not covalently bound to components of the cell and will thus slowly diffuse out of cells after fixation, gradually staining all cells as dead. 

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.

There are two easy options. One is to heat-inactivate the cells by placing at 60°C for 20 minutes. The second is to subject the cells to 70% ethanol. Alcohol-fixed cells can be stored indefinitely in the freezer until use, potentially up to several years.

1. Centrifuge cells, pellet, and remove supernatant. 
2. Fix cells: Add 10 mL ice cold 70% ETOH to a 15 mL tube containing the cell pellet, adding dropwise at first while vortexing, mix well. 
3. Store in freezer until use.
4. When ready to use, wash twice and resuspend in buffer of choice.

We have validated the following kits for use on the Countess™ II FL Automated Cell Counter:

• LIVE/DEAD™ Viabilty/Cytoxicity Kit (Cat. No. L3224) containing calcein AM and ethidium homodimer-1
• ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Cat. No. R37609)
• ReadyProbes™ Cell Viability Imaging Kit, Blue/Red (Cat. No. R37610) containing NucBlue Live/NucGreen Dead and NucBlue Live/propidium iodide

See this Application Note for details.

The Image-iT Red Hypoxia Reagent has an approximate Ex/Em maxima at 490/610 nm. You may use a 488 nm laser and a Texas Red emission filter. For lamp-based instruments, a YFP longpass filter set may be used (excitation range from 485 to 510 nm and emission from 550 nm up to 620 nm).

The Image-iT Green Hypoxia Reagent has an approximate Ex/Em maxima at 488/520 nm. We recommend using a 488 nm laser and a FITC/GFP emission filter.

Yes. We recommend using NucGreen Dead 488 ReadyProbes Reagent (Cat No. R37109) with Image-iT Red Hypoxia Reagent and NucRed Dead 647 ReadyProbes Reagent (Cat No. R37113) with Image-iT Green Hypoxia Reagent.

You may use cell-permeable nuclear counterstains or surface labels, but avoid any general cytoplasmic stains that may interfere with the emission of the hypoxia reagents.

Yes, they are the same reagent; the only difference is in the way these products are packaged. The Hypoxia Green Reagent for Flow Cytometry (Cat No. H20035) provides 2 vials, with 30 µg/vial. The Image-iT Green Hypoxia Reagent is shipped as either 1 or 5 vials with 840 µg/vial. Stock solutions for both products should be 1 mM in anhydrous DMSO.

The difference between these products is in the final working concentration of the reagent and incubation time. For flow cytometry applications, we recommend using a final concentration in the range of 0.5 to 1 µM with an incubation time ranging from 2 to 3 hrs. For imaging, we recommend a final concentration in the range of 1 to 10 µM with an incubation time ranging from 30 mins to 1 hr. For both applications, one should optimize the final working concentration and incubation time.

This could be due to storage of the product under anaerobic or low oxygen conditions. The Image-iT Green Hypoxia Reagent increases in fluorescence upon exposure to low oxygen environments, and this change is not reversible. For some ROS indicators, we recommend storing the reagent under dry nitrogen or argon to prevent oxidation during storage. However, this is not appropriate for the Image-iT Green Hypoxia Reagent.

Yes, as long as the fluorescent protein does not emit in the green range (˜510–535 nm). For cells that do not express a fluorescent protein, we recommend viewing unstained cells under the FITC channel to examine autofluorescence. For cells expressing a fluorescent protein, we recommend analyzing unstained cell samples to determine the extent the fluorescent protein emission may overlap in the FITC channel.

Yes, as long as the fluorescent protein does not emit in the red range (˜610 nm). For cells that do not express a fluorescent protein, we recommend viewing unstained cells under the TRITC and Texas Red channels to examine autofluorescence. For cells expressing a fluorescent protein, we recommend analyzing unstained cell samples to determine the extent the fluorescent protein emission may overlap in these channels.

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 and protocol. 

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.

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.

CHO-K1 cells were plated at 5,000 cells/well in a 384-well plate in the presence of a dilution series of staurosporine, in phenol red–free complete medium. The plate was incubated for 19 hours at 37° C/5% CO₂. PrestoBlue™ reagent and CellEvent™ reagent were added directly to the wells, and the plate was incubated for 30 minutes at 37°C/5% CO2 prior to reading the fluorescence. Florescence signal was detected on a Tecan Safire2 plate reader (bottom read) with settings of Ex 500 nm /Em 530 nm bandwidths of 7nm for the CellEvent™ reagent and Ex 560 nm/Em 590 nm bandwidths of 10 nm for the PrestoBlue™ reagent.

CellTrace™ Cell Proliferation reagents are all cell-permeant dyes that are cleaved by intracellular esterases to yield highly fluorescent compounds that also covalently bind to cellular amines, attaching the dye to various cellular components and providing a very stable signal. These reagents show little cytotoxicity with minimal observed effects on the proliferative ability of many cells. 

We provide the CellTrace™ reagents in small aliquots and strongly recommend discarding any unused DMSO dye stocks. The CellTrace™ reagents have diacetate groups to cap the charges on the dyes to make them cell permeant and succinimidyl ester amine-reactive groups for long-term retention. Both diacetates and succinimidyl esters will readily hydrolyze if any water is present during storage. DMSO is hygroscopic and thus readily absorbs water from the atmosphere. If you must store your dye stocks, you will need to use a good quality, anhydrous DMSO stock that has not been opened often and seal the vials and place in an air-tight containing some desiccant to keep the DMSO/dye stock solution as dry as possible. Store at –20°C. Use within a short time period.

If you are using EdU or EU, the simplest thing to do is purchase a complete kit that is specific for the type of assay you are performing, either flow, imaging, or a microplate assay. The complete kits contain the azide detection reagent as well as copper and the buffers necessary to perform the click reaction. If you have a different alkyne metabolite analog or do not wish to purchase EdU or EU, then you will need to purchase any of our azide dye detection reagents and the Click-iT™ Cell Reaction Buffer Kit (Cat. No. C10269), which contains the copper solution and the other buffer reagents necessary to perform the click reaction for flow and imaging assays. If you wish to perform a microplate assay, then you are limited to the Click-iT™ EdU Microplate Assay (Cat. No. C10214), which provides all the reagents necessary for the click reaction as well as the reagents necessary to perform the amplification of the signal so that it can be detected on a microplate reader.

Unfortunately, the actual concentration of the azide dyes used in the Click-iT™ assay kits is proprietary, so we cannot provide the exact concentration used. For a general guideline, we recommend making up a 1–10 mM DMSO stock solution and using at approximately 1–10 μM final concentration in the click reaction. Too high of a dye concentration can result in high nonspecific background or dye aggregates; too low of a concentration may not give a strong enough signal.

The copper sulfate solution component supplied in the Click-iT™ kits is 100 mM copper sulfate in water. Copper (II) sulfate (CuSO₄) powder is available from many chemical suppliers. 

The Click-iT™ Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the incorporated alkyne group and thus minimizes copper damage to biomolecules. The original Click-iT™ kits use an unmodified azide dye and higher copper concentrations to perform the click reaction, which may inactivate enzymes, including HRP, and will quench the fluorescence of GFP, RFP, mCherry and other fluorescent proteins, as well as R-phycoerythrin. If you do not wish to modify your antibody staining protocol or have fluorescent protein–expressing cells, then use the Click-iT™ Plus kits.

No, the detection reagent and reagents necessary to perform the click reaction cannot be intermixed between the Click-iT™ Plus and original Click-iT™ kits. The Click-iT™ Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the click reaction, while the original Click-iT™ kits use an unmodified azide dye and higher copper concentrations to perform the click reaction. 

The Click-iT™ EdU flow kits use a lower azide dye concentration than what is optimal for the EdU imaging kits, so it is not possible to use a flow kit to label cells for an imaging experiment. It is possible to use a Click-iT™ EdU imaging kit for samples for flow analysis, but it would be necessary to optimize the azide dye concentration first. The azide dye concentrations used in the Click-iT™ EdU kits are proprietary.

Yes, you can store samples after fixing in formaldehyde and washing, before the permeabilization step. Just keep the cells in PBS, cover and seal the container well, and store at 4°C. The cells should be fine for at least a week. You can also store the samples after the click reaction and wash steps and then perform any immunostaining and nuclear counterstaining on the following day.

Yes, EdU is frequently used for in vivo assays and acceptable EdU incorporation can be obtained following injection or media incubation. Optimal EdU incubation times and concentrations will depend on the incorporation method and the organism/tissue type. Recommendations on EdU incorporation methods in various model organisms and protocols for detection of EdU in tissue samples can be found in the In Vivo Use of Click-iT EdU Cell Proliferation Assays application note and the Click-iT EdU Labeling In Vivo Cell Proliferation Protocol.

Table 2 of the EdU (5-ethynyl-2'-deoxyuridine) product manual also provides some recommendations. Methods that have been optimized for BrdU labeling for your organism are a good starting point. Tissue samples can be frozen or paraffin-embedded and then processed using normal procedures for subsequent IHC staining. For detection of EdU, use any desired method for fixation and permeabilization, and then perform the click detection reaction exactly according to the protocol for cultured cells in the product manual.

We have not validated the use of EdU for proliferation in 3D culture systems, but as this reagent is compatible for labeling cells in vivo, it is also expected to label cells in 3D culture systems. There are a number of reports in the literature that use this product in 3D culture systems; here are some citation:

Robertson FM, Ogasawara MA, Ye Z et al. (2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype. J Biomol Screen 15:820–829.

Yes, EdU and BrdU labeling can be combined for dual-pulse labeling of cell proliferation in cultured cells and in vivo. BrdU will be preferentially incorporated into DNA, so perform the EdU incubation first followed by the BrdU incubation. Removal of EdU from the media is not required in cultured cells when BrdU is added as the second label. Perform an alcohol fixation followed by some method of DNA denaturation as required for the BrdU detection protocol and then perform the click labeling reaction for detection of EdU followed by antibody labeling for detection of BrdU. Be sure to select a BrdU antibody that does not have cross-reactivity to EdU, such as our MoBU-1 clone (Cat. No. B35141). Many BrdU antibodies have been shown to have some amount of cross-reactivity with incorporated EdU. Here is a link to an example protocol for dual-pulse labeling using EdU and BrdU.

It is possible, but if you have not completely labeled all of the metabolically incorporated EdU in the first click reaction, then it will be labeled in the second click reaction for TUNEL labeling, leading to false positives for apoptotic cells. It would be simpler to combine Click-iT™ EdU labeling with BrdU TUNEL labeling, as BrdU detection will not cross-react with EdU labeled cells. If you really wish to perform a double EdU labeling for both proliferation and apoptosis detection, then you should repeat the click reaction to detect the metabolically incorporated EdU using fresh click reagents to ensure that all of the incorporated EdU is labeled before performing the EdU TUNEL assay. You should then perform a control no–TdT enzyme EdU TUNEL assay to verify that there is no signal generated with the TUNEL click reaction.

No, the EdU metabolic labeling reagent must be used on live cells, but the actual click detection reaction must be performed on fixed and permeabilized samples, as the azide detection reagents and buffer components are cell impermeant.

Yes, but we have not optimized the amount of substrate to use. The labeling may be punctate and therefore an intense dark label such as DAB is recommended.

You need to only substitute the peroxidase-conjugated streptavidin with a dye-conjugated streptavidin and not use the DAB. You would have to optimize the final concentration of the dye-conjugated streptavidin to use.

Annexin V staining is best analyzed on live cells. If you need to fix your cells for analysis, then fix in 3.7% formaldehyde in PBS containing calcium and magnesium to maintain binding during fixation. The signal will not be retained after permeabilization, thus annexin V staining is not compatible with internal antibody labeling. 

Trypsinize first and then allow the cells to recover about 30 minutes in optimal cell culture conditions and medium before staining with annexin V conjugates. Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing for annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. For lightly adherent cell lines such as HeLa and NIH 3T3, you could use a less harsh (non-enzymatic) dissociation product like Gibco™ Cell Dissociation Buffer (Cat. No. 13151014).

Annexin V staining is not typically used in imaging experiments; it is a better reagent for flow cytometry analysis. All cells will stain to some extent, so it can be difficult to distinguish a relatively bright annexin V–stained cell from a dimmer non-apoptotic cell. Caspase activation, detected using our CellEvent™ Caspase 3/7 or Image-iT™ LIVE Caspase detection kits, is a better method for detecting apoptosis in an imaging assay.

For imaging applications, we recommend fixing with 3.7% formaldehyde at room temperature for 15 mins. After fixation, cells can be imaged for up to 24 hrs, but the signal will gradually degrade over time. For flow cytometry applications, fixation is not recommended.

CellEvent™ Caspase 3/7 is the only reagent that can be used to detect apoptosis in live cells without washing. The reagent is nontoxic to cells and will only become fluorescent upon binding to DNA after caspase activation. 

You should make an apoptotic cell control and also a dead cell control. Common reagents for used for generating apoptotic control cells are staurosporine, camptothecin, and valinomycin. Other general drug treatments for generating apoptotic control cells are listed in the table below. Not every agent will induce apoptosis in every cell type, and the concentration and incubation time used may need to be optimized. Depending on the agent chosen and the concentration used, maximal induction of a particular protein may occur at any time between 8–72 hours post-treatment. 

There are two easy options for generating dead cell controls. One is to heat-inactivate the cells by placing at 60°C for 20 minutes. The second is to subject cells to 70% ethanol (see protocol below). Alcohol-fixed cells can be stored indefinitely in the freezer until use, potentially up to several years. 

1. Centrifuge cells, pellet, and remove supernatant. 
2. Fix cells: Add 10 mL ice-cold 70% ETOH to a 15 mL tube containing the cell pellet, adding dropwise at first while vortexing, mix well. 
3.  Store in freezer until use.
4. When ready to use, wash twice and resuspend in buffer of choice.

We have validated CellEvent™ Caspase 3/7 Green Detection Reagent (Cat. No. C10423) in combination with SYTOX™ Red Dead Cell Stain (Cat. No. S34859) for discriminating apoptotic cells from dead cells using the Countess™ II FL Automated Cell Counter. For further details, please see this Application Note. 

See Table 1 in this article where a Jurkat model system was induced with 10 µM camptothecin for time periods of 0 to 4 hours. It is important to note that these results were studied using a single cell type and induction system; results may differ for other experimental systems. Increased membrane permeability in apoptotic cells can be discriminated from dead cells using YO-PRO™-1 dye in combination with propidium iodide or the SYTOX™ dead cell indicators. Other mid-apoptotic events are increased ROS production (detected with CellROX™ reagents, H₂DCFDA), changes in cellular pH (BCECF, SNARF™-1) and calcium release (Fluo-4, Fura-2, Indo-1). No single parameter defines apoptosis under any condition, so it is best to employ a multi-parametric approach when studying apoptosis.

The original Click-iT™ TUNEL assay kits were optimized for cell culture samples and may also be used on tissue samples. We recommend using the Click-iT™ Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT™ Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue. The protocol was modified to improve accessibility of the TdT enzyme into the multiple cell layers of tissue samples. In addition, we found that the original Click-iT™ TUNEL kit may show higher non-specific binding and punctate staining in tissues compared to the Click-iT™ Plus TUNEL kit. One method that works well to increase permeability for tissues is to replace the detergent permeabilization step with proteinase K digestion and then refix the sample in formaldehye. The Tissue Fixation and Permeabilization protocol in section 3 of the Click-iT™ Plus TUNEL kit manual can be followed for tissue samples using the original Click-iT™ TUNEL assay. Pepsin and other proteolytic enzymes can also be used to improve permeability.

We have validated the Click-iT™ Plus TUNEL for In Situ Apoptosis Detection assay on 5–20 µM thick FFPE sections of mouse intestine, kidney, liver, heart, and colon.

Most tissue samples will be adequately digested in 15 minutes. The optimal incubation time will vary depending on tissue type and thickness. We have observed that brain tissue needs longer proteinase K treatment than other tissues tested. 

We have not validated the use of EdU TUNEL for apoptosis detection in 3D culture systems, but as this reagent is compatible for labeling cells in vivo, it is also expected to label cells in 3D culture systems. There are a number of reports in the literature that use this product in 3D culture systems; here are some citations:

Robertson FM, Ogasawara MA, Ye Z et al. (2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype. J Biomol Screen 15:820–829.

Yes, additional Terminal Deoxynucleotidyl Transferase (rTdT) can be purchased as Cat. No. 10533-065 or 10533-075. Additional TdT reaction buffer can be purchased as Cat. No. 16314-015.

Yes, other colorimetric stains can be used.

Yes, but we have not optimized its use with this kit.