Hi all,
Has anyone compared the live cell imaging media sold by Sigma with Invitrogen’s Live Cell Imaging Solution or Gibco’s FluoroBrite DMEM?
Are there alternatives to consider?
Thanks in advance,
Will
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Hi,
I have tried Gibco DMEM (without phenol-red, with HEPES) and that works fine for long term live cell imaging.
Thank you for your response. I’ve also used the same medium for <6 hour live-imaging sessions.
I guess my main concern is the presence of riboflavin and other components which induce cytoxicity during imaging. Sometimes stressed cells are easy to see by eye (hello morphological signs), but there may be more subtle effects like this.
No, I haven’t directly compared them. I have used different media from different sources for live cell imaging that are phenol red free (and sometimes riboflavin free). I’ve used CellGro, Gibco, Sigma, they are all good. Double check the formulation to be sure they are complete, sometimes glutamine isn’t included in the phenol red free medium, for example. Live Cell Imaging Solution is simply that - a buffered solution of salts and HEPEs, that keeps cells happy for a few hours (4 or so), it is not a full culturing medium designed for culturing cells - I have used it, and it had no noticeable fluorescence, fluorescent noise of cells growing in a glass bottom dish on the microscope was very low. OptiMEM can also be used, it seems to be less fluorescent than normal growth medium. Some people also ween their cell lines down to 5% serum as serum contributes some fluorescence. It depends on your application, whether you need a complete culture medium, or if a solution is ok for you. You could make Live Cell Imaging Solution yourself, the website indicates what’s in it. Even DBPS with 20mM HEPEs, pH 7.4 is good (if only need an hour or so). Good luck!
Adding to my previous comments. Phenol red free, riboflavin free medium can be VERY expensive (has to do with ordering a production run that doesn’t contain the riboflavin), maybe there is a vendor now that cells it, but when I asked a few years ago it was >$500 per 500ml bottle. Not sure if you need these hints but they are good to hear again: In short experiments, as long as you have a good chamber that keeps the environment correct and there is no evaporation (this is important and some cell lines are very sensitive to this), then in my experience, phototoxicity is the biggest issue at causing stress, using a non-florescent medium, allowing you to minimize light intensity in particular, and also exposure time, may help. Intensity is a real problem, if you can get away with binning the camera then you can use even less intensity. What kind of microscope are you imaging on? If its a LSCM then, if appropriate you can slightly open the pinhole and drop the power (there are many more hints). Good luck (again).
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Interesting, thank you for the insight regarding the cost of phenol red free and riboflavin free media as well as your other comments.
I really should have provided additional context in my original post; the two main reasons are
overnight/multi-day and super-res imaging. We’ve got a good variety of scopes in our core, and I’d hate to think that riboflavin-and-friends means that our users can’t take full advantage of multi-point acquisitions.
Here’s one example where I’d like to know if I should worry about ROS generation in media: On a lattice light sheet microscope, how long can I keep using the same 3 or 12mL of media for different cells on the same coverslip? It’d be mighty convenient to continue acquisitions remotely. @talley, do you swap for fresh media every time you change coverslips?
Since you’re dropping info for future readers, shout out to #1 and #2 for being great reads!
I’m having a tough time getting over how “avoid riboflavin” shows up in imaging guides and yet seems to be ignored most of the time. But people love saturating images, so…
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To be clear, medium that is both phenol red and riboflavin free is what I found to be very expensive.
I’m not to familiar with long timelapses using light sheet. 12 ml for a single slip is a lot of medium, I’d like to think you could go quite long, is perfusion on option? Maybe not on a light sheet given some of the challenges it presents.
One thing to remember is the biology - there are cells that probably exhaust their media or acidify or release something else that is toxic, where other cells do not. 3 and 4 day time lapses are pretty common and these cells are often in 6 or
12 well plates. You can add ROS scavengers to medium to help address if effects are due to ROS production, an old school scampered is ascorbic acid, but I’m sure there are other, better options.
Best wishes on your experiments.