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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I am storing the DMSO stock solutions of the CellTrace cell proliferation reagents and am not obtaining a good signal with my cells compared to freshly dissolved dye stock. Why does the product not work after storage?
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
I am using the CellTrace Cell Proliferation reagents and am not obtaining good separation of my cell generation peaks. How can I improve the peak separation?
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.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Can I store the stock solutions of the CellTrace reagents and how do you recommend storing them?
We provide the CellTrace reagents in small aliquots and strongly recommend discarding any unused DMSO dye stocks. Some of the CellTrace reagents have diacetate groups to cap the charges on the dyes to make them cell permeant, and some have succinimidyl ester amine-reactive groups for long-term cellular retention. Both diacetates and succinimidyl esters will readily hydrolyze if any water is present during storage. DMSO is very hygroscopic and thus readily absorbs water from the atmosphere. We do not recommend storing stock solutions of CellTrace reagents because storage of the product in solution will inevitably lead to partial or complete loss of reactivity.
