Molecular Probes™ have developed a number of membrane permeable fluorescent probes that can be used to visualize live cells. The CellTracker™ Probes are fluorescent chloromethyl derivatives, which are processed into membrane impermeable compounds that are retained inside the cells up to 72 hours after loading, and can be passed to daughter cells. The probes contain amino groups so can be cross-linked using formaldehyde or glutaraldehyde for sample storage and analysis. These products have a variety of applications ranging from cell viability assays, cell adhesion, cell migration and cell-cell fusion studies.
A range of probes are available, from CellTracker™ Red to several different formulations of CellTracker™ Blue, Green and Orange which differ slightly in chemical structure, fluorescence activation and emission spectra – the range of spectra means dual labeling is possible for some dye combinations. The probes are supplied as desiccated samples stored at -20C. These are made into 10 mM working solutions in DMSO which are also stored at -20C; both forms are stable for 6 months. While DMSO is toxic to cells, the stock solution is well diluted, so effects on cell health should be minimal. If working with sensitive cell lines, it might be a good idea to test the toxicity of DMSO on cells before starting to label.
The protocol for labeling cells is quite simple: starting with a healthy culture of the cell line you wish to label, dilute the dye from the 10 mM DMSO stock to the desired working concentration using serum-free media. The optimal concentration of dye for labeling is best determined for each cell line separately, but according to the manufacturer, it is usually in the range of 5-25 µM. To load the cells, simply remove the growth media, and incubate in the dye-containing media for 15-45 minutes. Again, the optimal incubation time for incorporation is best determined using the cell line of interest. Once loaded, the media on the cells can be replaced with normal growth medium. Cells should be incubated for another 30 minutes to ensure modifications to the dye can take place. After this, cells can be incubated further, or fixed and stained for visualization under a microscope or a flow cytometer. Molecular Probes has a series of antibodies that bind to the probes, which can be used for additional enhancement of signal and visualization.
We have used some of the CellTracker™ Probes in our laboratory, and found them to be easy to handle and quite stable if stored correctly. We have labeled mostly mammalian cells and have found the dyes cause no visible toxic effects. In our cell lines (VERO, HEK293) the dyes were visible up to 24 hours after labeling. The dyes were analyzed by epiflourescent and confocal microscopy and showed bright fluorescence, distributed fairly evenly throughout the cytoplasm. The dyes were also able to be used to identify different cell populations via fluorescence activated cell sorting. Crosslinked sample should be kept in the dark at 4C for short term storage, or frozen at -20C to retain signal.