What is the approximate excitation and emission peaks of the CellTrace Far Red Proliferation reagent?
The approximate excitation and emission peaks of this product after hydrolysis are 630 nm and 661 nm, respectively. Cells labeled with CellTrace Far Red reagent can be visualized by fluorescence microscopy using standard Cy 5 filter sets or analyzed by flow cytometry in an instrument equipped with a 633/635 nm excitation source and APC channel.
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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 CellTrace Far Red dye be fixed with paraformaldehyde (PFA)?
Yes, the CellTrace Far Red dye can be fixed with paraformaldehyde (PFA). The dye covalently binds to cells and will not wash out after permeabilization or fixation.
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I would like to label two cell populations with two different CellTrace reagents and then co-culture these cells. Will the CellTrace reagent leave the cells to stain other cells?
We have not tested the use of the CellTrace reagents for co-culture applications. In theory, this may work, but you would have to test this on your cells of interest.
Find additional tips, troubleshooting help, and resources within our Cell Tracing and Tracking Support Center.