I am looking to assess the metabolic control of breast epithelial cancer cells in cancer-associated fibroblasts (CAFs) and the subsequent effect of this on doxorubicin chemoresistance using an in vivo mouse model. We have access to a stable GFP expressing fibroblast cell line, and are wanting to co-inject them into C57/BL6 mice with a stably transfected cancer cell line. So far one of the better options for us is using a mCherry plasmid (as this will not interfere with the GFP signal). However, as we are also making use of Dox in this setting, I am worried about the overlap in signal between mCherry and Dox auto-fluorescence. Is there perhaps a way of "compensating" for the signal when using confocal microscopy? If not is there a better fluorescent plasmid which will not overlap?
I would really appreciate any help with this.
The emission profiles for mCherry and Doxorubicin overlap substantially, and both have a peak emission around 600nm. You are right to be concerned.
Your choice of fluorescent protein will be dictated by the capabilities of your microscope, where you expect to the brightest and dimmest signals to come from, and what you hope to achieve from the experiment. If you have a HeNe laser, you can consider a far-red protein like E2 Crimson. If you have a multiline argon with 458nm, you can look at a blue protein like AmCyan. You can use a violet laser (405nm) to excite blue proteins, but I wouldn’t recommend this if you are hoping to do live-imaging, and you also need to be mindful of bleaching with a 405. If you are just going to look at sections then a 405 will work (bleaching issues are overcome by sequential scanning, with the 405 being the 'last' laser).
The blue proteins tend to be a bit of a pain to use with GFP because of spectral overlap issues (they are much better with YFP), but sequential scanning can usually overcome this. This is also less of an issue in your proposed experiments, because the Cyan and GFP would be coming from separate cells, so you shouldn’t have any troubles discriminating fibroblasts from cancer cells.
Generally-speaking, the far-red proteins are better for in vivo work, and I’m sure you could discriminate a Crimson+ cancer cell from surrounding doxorubicin+ cells (unless the doxorubicin signal is very bright and the Far-redFP/Crimson expression is weak). But if you are hoping to actually measure the doxorubicin, then I would not recommend you use any fluorescent protein that may interfere with these measurements.
Good luck!
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