I am trying to visualise actin by lifeact GFP by perkin elmer spinning disc microscope,but for some reason my transfected cells are dying. I have added 100uM HEPES in the media and using temperature controller chamber. I can not figure out what is wrong. It will be great if I can get get any suggestions about it.
Just to clarify - Are your cells dying while on the microscope stage?
If so, this could be the result of phototoxicity, or levels of CO2. Phototoxicity would manifest as a gradual decrease in the viability of cells within the focal plane, and be directly related to excitation intensity.
However, my guess is that this has to do with CO2 levels and pH buffering. HEPES won't entirely eliminate the need for CO2 for long term imaging, so it might be good to include a flow of 5% CO2 into the chamber. The HEPES concentration can also be increased if you're having a problem with pH buffering; several live cell imaging solutions contain HEPES in the range of 10-2-10-3 M. You're on the low end of that range, so you might try a few more concentrated solutions when imaging.
Best of luck. Those lifeact:FP fusions can yield beautiful pictures.
Leave your cells on the stage with no imaging. Are they still dying? If so, then the problem is with your culture/incubation conditions as Brian suggested. If not, then you are killing them with over illumination. Taking z stacks at rapid rate with high laser power, for example, will stress cells. You can do this only for very short term time lapse. If you need long term time lapse, then use less laser power and space your acquisition intervals.
I've used CO2 independent media from Thermo for similar applications with good success.
Thanks a lot Brian and Christopher. As Brian suggested I increased the concentration of HEPES to 25mM and it worked.
Hi, Ravindra.
In addition to the solid suggestions above, try to avoid any unnecessary UV exposure immediately after you record the images. Try focus on the cells under bright field, quick look and focus the actin filaments in detail. By doing so you will be adapting yourself to manipulate filter block switch-over and blocking the UV. Actin is 20~25 % of the cellular proteins. When you excite the abundant fluorescent molecules, damage to the cells is great. If you put the dish back into CO2 incubator for ~30 min, unploymerized molecules will replace the exposed molecules through dynamic nature of actins.
Okay sure Sang Ho Lee .I will keep that in mind. Thank you.
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