Immediately after imaging our cells are fine and also up to about 12 hours later, then the health seems to rapidly deteriorate. Thoughts?


It is difficult to answer this question without knowing more details about your imaging setup. I will assume that you are imaging your cells in CO2-buffered cell culture media and that the environment is 37C, 5% CO2 and humidified. The gradual reduction in cell health suggests that one of the following may be occurring:

1. Media evaporation

Placing a vessel of sterile distilled water somewhere within the imaging chamber should help with media evaporation. If you are imaging in a multi-well vessel, I suggest also placing sterile water/PBS in all empty wells surrounding the wells containing your cultures.

2. Insufficient buffering of the media due to low CO2 levels

The system you are using may not be providing enough CO2 to your cultures for adequate maintenance of long term cell health. To test this, I would suggest placing your imaging culture in a media that contains phenol red and visually monitoring the color of the media over time. A change in the media color from reddish-orange to reddish-purple would indicate that there is likely poor CO2 gas exchange.

3. Phototoxicity due to light exposure

Some cell types are more sensitive to light exposure than others. It could be that your cells are becoming progressively more damaged with repeated light exposure over time. If this is the case, I would suggest that you image your cells in FluoroBrite DMEM or another media with low background fluorescence. The benefit of working with a low background medium is that you can reduce the intensity of potentially damaging fluorescence light without compromising the signal-to-noise ratio of your fluorophore. Another suggestion would be to give your cells a fresh supply of media prior to the point when you see health begin to deteriorate. This is because fluorescent light can cause the formation of free radicals in cell culture media which can be harmful to cells.

4. Toxicity due to overexpression of a fluorescently-labeled protein

The over-expression of a fluorescently-labeled protein can sometimes induce phototoxicity. You may want to consider cloning your protein into a TET-inducible vector or a vector with a weaker promoter such as EF-1 alpha. You could then perform clonal selection on a heterogeneous population of cells expressing this construct to identify a clone that expresses lower levels of your fusion protein which do not alter the phenotype of your cells.

We have an excellent team of highly knowledgeable technical service representatives at ThermoFisher Scientific. I suggest you reach out to Tech Services for further guidance if none of the ideas mentioned above helps to address your current problem.

This is the last question in the session.

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