Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support—Troubleshooting

Exposure to light may degrade the dye and these contaminants may promote precipitation. Trypan blue can also form orange/red fibrous aggregates if exposed to refrigeration or freezing temperatures. 

Unfortunately, there is no definitive answer to this question. The rate at which the cell-impermeant dye is absorbed depends on the cell type, their state of health, nourishment, engulfment activity, etc.  

Yes. alamarBlue™ reagent is stable to multiple freeze/thaw cycles. Be sure to warm the reagent in a 37°C water bath and mix it well to ensure a homogenous solution before use. 

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

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

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. 

We recommend increasing the incubation time of cells with alamarBlue™ reagent, changing the instrument’s gain or voltage setting, and checking the instrument filter/wavelength settings. Make sure to have positive controls (untreated, living cells) in the experimental design as a control.  

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

One reason could be that the dye in your alamarBlue™ reagent has precipitated, resulting in varying dye concentration. In such cases, the reagent should be warmed to 37°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. 

Yes. PrestoBlue™ reagent is 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. 

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

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

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. 

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.  

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

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°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. 

Here are some tips to obtain uniform staining and a bright, unstimulated parent generation peak:

• Dissolve the CellTrace™ dye stock immediately before use in the DMSO provided in the kit or in good quality, anhydrous DMSO to obtain the best reactivity and cell permeability.
• Stain in PBS or other amine-free, protein-free physiological buffer. Do not stain in medium.
• Start with a single-cell suspension and gently agitate the cells during staining.
• Quickly remove the unbound dye by incubating the cells in ice-cold media for 5 minutes and then wash twice more with pre-warmed media. 
• Include a dead-cell stain in the assay and gate only on live cells. 
• Analyze as many cells as possible from each sample.
• Use a low flow rate for analysis on hydrodynamic focusing cytometers. 
• A good staining concentration for the CellTrace™ dyes is generally within 1–10 µM, but the optimal concentration for a particular cell type will vary. Observe your cells in a stain dilution series to determine the optimal concentration for your cells.
• Some cell types may take up dye with a broad staining intensity distribution. If this is the case for your cells, then you will need to do an initial sort of the stained, unstimulated parent cells to select for a narrow peak distribution.  

We provide the CellTrace™ reagents in small aliquots and strongly recommend discarding any unused DMSO/dye stocks. The CellTrace™ reagents have acetyl groups to cap the charges on the dyes to make them cell permeant, and succinimidyl ester amine-reactive moiety to allow for covalent attachment to cellular components for long-term retention. Both acetyl groups 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 store the vial within an air-tight container containing some desiccant to keep the DMSO/dye stock solution anhydrous during storage. 

The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX™ Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT™ detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no–click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You can also perform the complete click reaction on a carrier solvent-only, no EdU or no-EU control to verify the specificity of the click reaction signal. 

• The click reaction is only effective when copper is in the appropriate valency. Except for the DIBO alkyne-azide reaction, azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest. 
• Do not use additive buffer that has turned yellow; it must be colorless to be active. 
• Cells need to be adequately fixed and permeabilized for the click reagents to have access to intracellular components that have incorporated the click substrate(s). 
• Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be oxidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction. 
• You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling. 
• Low signal can also be due to low incorporation of EdU, EU, or other click substrates. Other click substrates (e.g., AHA, HPG, palmitic acid, azide, etc.) incorporated into cellular components may have been lost if not adequately cross-linked in place or if the wrong fixative was used. For click substrates that are incorporated into the membrane or lipids, you should avoid the use of alcohol or acetone fixatives and permeabilizing agents. 
• The incorporated click substrate must be accessible at the time of the click reaction; labeling of incorporated amino acid analogs may be lower in native proteins relative to denatured proteins. 
• You may need to optimize the metabolic labeling conditions including analog incubation time or concentration. Cells that are healthy, not too high of a passage number and not too crowded may incorporate the analog better. You may create a positive control by including extra doses of the click substrate during multiple time points during an incubation time that spans or closely spans the doubling time of the cell type of interest. 

The copper in the click reaction denatures DNA to a small extent (although not as much as is required for efficient BrdU detection), which can affect the binding affinity of DNA dyes including DAPI and Hoechst™ stain. This effect should only be apparent with the classic EdU kits and not the Click-iT™ Plus EdU kits, which use a lower copper concentration. 

Trypsinization or mechanical scraping of cells temporarily disrupts the plasma membrane, allowing annexin V to bind phosphatidylserine on the cytoplasmic surface of the cell membrane and thus leading to false positive staining. Allow the cells to recover for about 30 minutes in optimal cell culture conditions and medium after trypsinizing/scraping so that they can recover their membrane integrity before staining. For lightly adherent cell lines, such as HeLa and NIH 3T3, another option is to use non-enzyme treatments like Gibco™ Cell Dissociation Buffer (Cat. No. 13151014).  

The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX™ Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT™ detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no–click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You should also perform the complete click reaction on a no-TdT enzyme control sample to verify the specificity of the click reaction signal. 

• The click reaction is only effective when copper is in the appropriate valency. Azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest. 
• Do not use additive buffer that has turned yellow; it must be colorless to be active. 
• Cells need to be adequately fixed and permeabilized for the TdT enzyme and click reagents to have access to the nucleus. Tissue samples require digestion with proteinase K or other proteolytic enzymes for sufficient TdT access.
• Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be o xidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction. 
• You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling. 
• Your cells may not be apoptotic. Prepare a DNase I–treated positive control to verify that the TdT enzymatic reaction and click labeling reaction are working correctly.