CellTracker™ Deep Red Dye, 20 x 15 μg - FAQs

Calcein, AM and FDA (fluorescein diaceate) are examples of some dyes used for this application. Since these dyes are not incorporated or covalently attached to any cellular components, they may have a short retention time as some cell types may actively efflux the dye out of the cells. The CellTracker and CellTrace dyes include either a mild thiol-reactive chloromethyl group or amine-reactive succinnimidyl ester group to allow for covalent binding to cellular components, providing for better retention. As with any reagent, one should empirically determine retention times for the cell type used.

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Calcein, AM is a good choice for cell tracking and as a general cytoplasmic stain. However, it doesn't bind to anything and may be actively pumped out of the cells within a couple hours, which is likely what happened. The retention of Calcein within live cells is dependent upon the inherent properties of the cell type and culture conditions.

For long-term imaging, you may wish to consider a reactive cytoplasmic stains such as CFDA, SE or the CellTracker and CellTrace dyes.

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Yes, the CellTracker dyes react with any accessible thiol part of the protein and can be fixed. However, some CellTracker dyes may be attached to small metabolites that can leak from the cell following permeabilization. This can result in decreased fluorescence.

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The retention of signal is not based on time, but rather on the rate of cell division over time and, for some cell types, on the rate of active efflux, protein turnover or other metabolic events. Dependent upon the cell type, it may be possible to see signal that lasts far beyond 72 hours. On the other hand, it may also disappear sooner than 72 hours.

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The CellTracker reagents have a reactive group that will allow covalent binding to various cellular components, large and small. When bound to small cellular components, if these are not cross-linked upon fixation, they may leach out, hence a loss of some signal.

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With excitation and emission maxima of 630/650 nm, any standard Cy5 or Alexa Fluor 647 filter set may be used. The reagent is efficiently excited using a 633 or 635 nm laser.

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One possibility is that there is spectral bleedthrough between the dyes. Be sure to check the single-color samples by imaging the red cells in green and imaging the green cells in red, using the optimal imaging settings for the other color. If you see bleedthrough with these controls, then you will have to reduce the dye label concentration to reduce the brightness of the dyes, or choose dyes that are farther apart spectrally. If the issue isn’t bleedthrough, another possibility is that the cells were not adequately washed after staining, allowing some unincorporated dye to remain and label the other cells after they were introduced. Extending washes and wash times should help with this.

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First, make sure you aren’t staining in the presence of serum, since serum can have esterase activity that can prematurely cleave the AM group on these dyes, preventing entry into cells. After staining, it’s okay to return the cells to medium containing serum. After this, you can try increasing the concentration and label time to get a higher intensity.

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Calcein dyes diffuse into cells, the 'AM' moiety is cleaved by cellular esterases and then are observed in the cytoplasm without binding to anything. This provides a 'whole cell' label. Calcein dyes may be pumped out by normal cellular efflux mechanisms, sometimes within a very short time, especially for cell types that may exhibit drug resistance, unless the efflux is inhibited (such as with probenecid). The dyes are not crosslinked with aldehyde-based fixation, unlike protein-binding CellTracker dyes, and thus will be lost upon fixation. Additionally, any disruption of plasma membrane, such as with detergents or trypsinization, will lead to leakage of the dyes from the cell.

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This is not recommended. When these stains bind to DNA and RNA, they may affect the normal function of the nucleic acids, disrupting transcription, as well as replication. Other reagents, such as CellTracker dyes or Qtracker reagents are more optimized for tracking without disrupting normal activity. If a nuclear label is still desired, though, and the cells are mammalian and non-hematopoietic, CellLight nuclear reagents can transiently transfect cells to express GFP or RFP on a nuclear-expressing protein for up to several days without affecting function.

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Please see this Web link (http://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html) to help you choose the right option for your application. Start by planning how long you want to track your cells, then consider the mechanism of binding. Calcein dyes are very uniform in label and are good for short-term cell migration, but may be rapidly effluxed from some cell types. Lipophilic cyanine dyes, such as DiI, DiO, and similar dyes label cell membranes, don’t disrupt function, and can last longer, but have the potential to cross to other cells if membranes fuse. They are also lost upon permeabilization. CellTracker dyes are better for longer-term labeling, as they possess a mildly reactive chloromethyl moiety that allows covalent binding to cellular components. CFDA SE also covalently binds to cellular components. With all the reagents, their retention within cells is dependent upon the rate of cell division and the inherent properties of the cell (active efflux, membrane and protein turnover rates, etc.) and reagents that allow for covalent attachment exhibit longer retention than those that do not.

The longest-lasting and brightest options are the Qtracker reagents, which are taken up through endocytosis. These are so bright individual quantum dots can be detected, and are also robust enough to survive not only fixation and permeabilization, but even the heat and solvents used in paraffin processing.

Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.