Hello everyone,
I have a question concerning the CellTracker Red fluorescence dye. Lately, our cells are dying when stained with CTR and then live imaged (we do timelapses over several hours, imaging ever 10-30 minutes). All the controls (stained, but not imaged and vice versa) seem fine, so it is (so far) just the combination of staining and prolonged imaging which kills the cells. As a final working concentration, the cells recieve 11 µM CTR for 45 min at 37 °C.
We are thinking of testing another dye to see if the fault lies with the imaging itself but that has not arrived yet.
Does anyone have any experience working with CTR and come across similar problems? Any suggestions would be welcome!
Hi. I'm with Thermo Fisher Tech Support. I've also used that dye and other CellTracker dyes when I was in R&D here.
11uM is probably a fine concentration, but 45 minutes seems like a long label time to me. Overlabeling (either too high a concentration and/or too long label time) can lead to toxicity effects. Your labeled, but unimaged, control looks fine, but remember that when you are imaging labeled samples, photobleaching occurs, particularly if you image over a long time, which creates free radicals and singlet oxygen that leads to increased cell toxicity. Some cell lines are more sensitive to others. Another thing to note is the binding mechanism. This dye has a chloromethyl functional group that binds to free thiols on proteins, particularly glutathione, in the cytoplasm. This, too, can lead to effects on cell functionality.
So here are some things to try:
1) reduce your label time and/or concentration, as long as your initial intensity is still sufficient for imaging.
2) reduce photobleaching effects by reducing your light exposure and compensating by increased gain or exposure time settings, reduce the frequency of imaging, or using ProLong Live (a live-cell antifade solution we sell).
3) Try a different dye. We have other colors of CellTrackers. Contact tech support ([email protected]) and we can help you choose one and offer a free goodwill replacement with it if you ordered within the past year. Feel free to mention my name. They'll need the catalog and lot number, order date, and any order numbers for your CellTracker Red. Since you are in Germany, our European tech support team will need to process it for you.
Cheers, Jason
Hello Marc-Thorben Knoll ,
You might want to check this fluorescent dye for life-cell imaging:
https://luminicell.com/products/cellaris-670-cell-labelling-kit-red
Cellaris is an AIE dye specifically designed for live-cell imaging. It is highly biocompatible and stable, providing long-term fluorescence for up to 10 cell generations in vitro!
Hope this helps with your research 😊
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