Dear Friends,
I would like to know if anyone have used any fluorescent dye that can stain the live cells in culture? I want to stain two different types of cell lines with different (colored) dye and want to study their interaction in a specific environment. I am going to use a live cell imaging platform and it would be a 72-96 hrs long experiment. I have previously used the Qtracker™ staining, but they are toxic for my cells. Now, I am looking for non-toxic stains. Any suggestion will be very helpful for me.
Thank you
Dear Purusottam,
if your cells are in culture and can be transfected, you could use overexpression of tagged proteins to follow the two different cells, e.g. one expresses GFP the other RFP (green- and red fluorescent proteins, respectively). if photobleaching becomes a problem, you could also express a protein that contains a SNAP tag and label it with more photostable dyes such as SNAP-Cell Oregon green ( https://www.neb.com/products/s9104-snap-cell-oregon-green#Product%20Information).
Calcein based dyes could be also of use. check for this the following thread on RG: https://www.researchgate.net/post/What_is_a_suitable_non-toxic_cell_permeable_dye_to_mark_cytoplasm_of_living_neurons
good luck
Darius
Dear Purusottam,
I agree, Calcein based dyes are a good solution. For living cells only ( https://www.thermofisher.com/order/catalog/product/C3100MP )
I also recommanded to use CFSE Dye or VPD dye. For both, the intensity of the fluorescence will decrease through cell proliferation. You will be able to follow fluorescence through several days/week ( https://www.thermofisher.com/order/catalog/product/C34554 ) ( http://www.bdbiosciences.com/ds/pm/tds/562158.pdf )
Hope it will help
Best
Adrien
Hello, Purusottam.
Yes, Darius and Adrien gave the answer. Dyes may survive a few cell cycles, gradually deteriorate in the cells. Make your own GFP cell lines by selecting trasfectants in drug. They will persist generation after generation. Even after photo-bleaching by UV, the GFP will recover from a simple transcription and translation, leading to ever-glowing cells within a few hours back into the incubator.
Hope you challenge it.
Dear all, Thanks a lot for your answers. My cell lines are drug resistant cancer cells that I have made in the lab from the parental sensitivity cells. I fear that trafection might affect their protein expressions specially my protein of interest and their migratory and invasive property. What are your suggestions on this? Therefore, I am looking a dye but I am not sure if this also will effect my protein of interest and the cellular functions like migration and invasion. Please share your experiences. It would definitely help me!
Hi, Purusottam.
According to our experience on MCF7-GAPDH-RFP and MDA-MB-231-GAPDH-RFP mutant cell lines, doxorubicin, azacytidine, sodium butylate, VPA and other epigenetics-disturbing drugs have been used along with wild types of MCF7 and MDA-MB-231 cells. Similar results in cell behaviour, proliferation, asymmetric cell division, modification into heterotypic cells were obtained. You only treat for ~7 days increasing levels of G418 for selection. That's all. You still have WT cells along with the transfectants.
Dear Purusottam,
I've used the following with great success. I ran a few optimization experiments to ensure non-toxicity and compared by western blotting the protein levels of stained cells to non-stained cells. Hope this helps!
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-tracing-tracking-and-morphology/cell-tracking.html#prd
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