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Having difficulties with your experiment?
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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:
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 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.
For Research Use Only. Not for use in diagnostic procedures.