I am about to conduct a pilot experiment to detect resting sarcoplasmic reticular, and cytosolic calcium levels in primary murine myotubes (differentiated for days 4 and 7), under resting conditions and using Thapsigargin (to inhibit SERCA, and empty out SR calcium). I wanted to use either mag-fluo-4/AM or mag-fluo-5N (green) in conjunction with Rhod-3/AM (orange/red) for detection by microscopy. I do understand that these two have different Kd values for calcium. Could you please recommend which dye to use in this case.
Thank you!
Hi Viraj,
I'm attaching a list of papers that you might find useful in your search. The first one, to my point of view, is the most important for you. In that one Zhou et al used Mag-Indo-1 to check intra SR calcium. A couple of good things about working with this dye are, its high kd and also its ratiometric properties. The problem of Mag-Indo is that you need to excite it with UV and for some people is not possible for technical reasons. That is why I am also pasting a couple of articles, one of them is mine and I used Fluo-5N but in skeletal muscle fibers and the other one was performed in cardiac muscle with Mag-Fluo. As you can see, any low affinity dye might be good for you, but if you are not able to use Mag-Indo, I would choose fluo-5N (only due to the dynamic range of it).
http://ajpcell.physiology.org/content/290/2/C539.full.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687039/
http://onlinelibrary.wiley.com/doi/10.1113/jphysiol.2012.243444/abstract
Let me know how it works and good luck!
Carlo
I’ve tried Fluo-5N, which works very well in rabbit hearts (http://circres.ahajournals.org/content/114/9/1410.long), but no success in perfused mouse hearts. Mag-fluo-4 does work in mouse hearts; it’s a bit tricky, though. The loading of the Mag-fluo4 requires room temperature. When the heart is warmed up after loading, the dye initially detects cytosolic Ca, not SR. After about 10-15 min, the cytosolic signal will disappear, and SR Ca signal shows up very nicely, which is consistent with what has already been reported - http://cardiovascres.oxfordjournals.org/content/85/4/671.long
Again, this is whole heart experiment.
Good luck.
Lianguo
Hello Lianguo and Viraj,
I just want to add that loading protocols are highly dependent of the type of cell you are using. Viraj, in the title of your question you wrote cardiac myocytes, however, in the body of your question you mentioned myotubes and this is why I recommended the first article (http://ajpcell.physiology.org/content/290/2/C539.full.pdf). If your cells are cardiac myocytes, I suggest that you follow the valuable help of Lianguo.
Again, good luck!
Carlo
Hi Carlo and Lianguo,
Firstly, thank you for your prompt replies and I apologize for the delay in getting back to you Carlo. I think there has been a slight confusion. I am working with skeletal muscle myocytes, and not cardiac myocytes, in cell culture conditions (although their uptake dynamics in the respective conditions might be quite similar, I suppose), but yes I do believe that the loading protocols are highly dependent on the cell type. I did find your suggestions quite helpful. I had already referred to your paper Carlo when you posted your reply. Thank you!
I did opt for using Fluo-5N/AM (Molecular Probes, Life Technologies), and am testing out using 5uM or 10uM for either 1 hour or 2 hours on a skeletal muscle cell line, loading in Ringers buffer.
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