I would like to do a time course experiment in C2C12 mouse myoblast cells over a ~7 day period. They will be transfected with GFP and I would like a nuclear fluorescent stain such as DAPI or Hoechst. DAPI requires fixing the cells (if i read correctly) but will the Hoechst dye allows the cells to continue expanding normally? Are there any better alternatives?
The alternative I come up with using Hoechst would be to plate a set of of cells for each timepoint and do the Hoechst stain at that time.
I think Hoechst33342 might be OK in terms of keeping cells live. But I think the fluorescence might not last that long. You may need to stain it again during the experiment. Why not you do a pre-experiment to test whether your cells are viable and fluroescent after 7 days? DAPI and some Hoechst might not be as good because they don't penetrate into cells as well if you don't permeabilize. You can also think about whether lentivirus encoding a nuclear localized GFP works for you. Some companies may sell those lentiviruses.
I agree with Xingmin Aaron Zhang, you should probably do a preliminary experiment to assure the cells are viable during that time and still fluorescent. Otherwise I guess you could set several experiments so that you can stain cells at each day.
In addition to the classical fluorescent dyes to mark nuclear DNA, there are a number of reporters that can be used to monitor cell proliferation over time. In my opinion, the best possibility is to use something that is stably expressed from the cell line and would not interfere with cell viability for sure. For instance, H2B-RFP or equivalent reporters could easily stain nuclear chromatin without any interference on cell cycle or cell divisions. If you want to be extremely precise in the analysis of cell proliferation, I would advice you the FUCCI system (http://www.lifetechnologies.com/it/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-cycle/live-cell-imaging-of-cell-cycle-and-division.html) to follow the different cell cycle phases by using complementary reporters.
Hoechst 33342 does work, but you'll need to titrate the concentration in your cells to find a good balance between staining and toxicity; you probably will loose the staining very quickly. You could use Draq5 or the Vybrant Dyecycle stains from Invitrogen - all are cell-permeable. I don't know how long any of these would last within the cells, but you could possibly re-apply as needed.
However, all DNA stains have some cytotoxicity and probably will influence the cell cycle in some way, so you may be best off following Stefano's advice.
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