I want to do a cell migration study for around 4 hours and need to fluorescently label the cells with a dye. What do you recommend?
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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I labeled my cells with Calcein, AM, but when I imaged the next day, there was no fluorescence from Calcein. Why?
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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Can the CellTracker dyes be fixed?
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.
Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.
Can the CellTracker probes last longer than 72 hours? Can the signal be lost in less than 72 hours?
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 Deep Red Dye signal on my live cells is quite robust, but signal drops after fixation/permeabilization. It is noted that this reagent is well-retained upon fixation, but why I am seeing a loss of some signal?
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.