I wanted to share some results we recently generated on the Seahorse Bioanalyzer, looking at the effect of GFP on cell metabolism. I was really surprised at the size of the response, and how reproducible it was. Hopefully others will find this data useful/interesting when interpreting their own results. Please let me know in the comments section below if you have found something similar.
This year my BSc(Hons) student (the very talented Ms Alex Webster) has been working on a C2C12 cell based project involving transient overexpression of a GFP tagged protein. Alex has completed several experiments so far looking at the effect of her protein on rates of proliferation and differentiation. Because of my ongoing interest in cell metabolism, Alex also used the Seahorse XF24 Bioanalyzer to examine the effect of her protein on OxPhos and glycolysis. She compared metabolism across three groups, control, GFP alone and her protein tagged with GFP, the results of the first two groups are presented below (we are planning on publishing a study with the tagged protein, so it isn’t included).
METHODS: The two graphs are from a single mitochondrial stress-test, run on a Seahorse XF24(3) machine, measuring oxygen consumption rate (OCR, top graph), and extracellular acidification rate (ECAR, bottom graph). In each graph, the green line/points indicate the GFP group, and black the control. Cells were seeded at a density of 25,000 cells/well and transfected with a commercially available GFP plasmid from Origene (pCMV6-AC-GFP) using Lipofectamine3000. The assay was run the next day as previously described by Dr David Nicholls (I will provide a full outline of our methodology in the ‘Muscle Methods’ section at a later date).
RESULTS: As you can see, overexpression of GFP in C2C12 cells results in a significant 3-fold increase in basal glycolysis (bottom graph) and a minor decrease in maximal oxidative capacity (peak observed between points B and C in top graph). Alex has repeated this experiment several times, and in every case experiment she has observed a 2-3 fold increase in glycolysis.
These results have important implications for the use of fluorescent tagging in cell culture studies. Altered cellular metabolism can lead to altered metabolite levels which can directly influence the transcriptional profile of the cell. Importantly, these results also indicate the need to confirm the metabolism of cells isolated in lineage tracing models (such as the often used ROSA26eYFP mouse).
As we have only used a single plasmid at this stage, I would be really interested to hear what experiences others have had when using fluorescent tags and the Seahorse.
Research in Myogenesis 
This is an important finding that has a huge and obvious implications and it further substantiates some other concerns about the impact of GFP on the subcellular localization of the tagged protein. Great science.Keep it up!
I have done some similar seahorse assays on C2C12s though with lentiviral transduction instead of transient transfection. Admittedly my comparisons all contained GFP (reporter was present in controls as well as with protein overexpression). I do get similar basal levels as your GFP in the mitochondrial stress assay, though don’t have a control without GFP for comparison.
Have you looked at the glycolysis stress test (it is just a shift from 0 glucose to high glucose (we used 10mM) and then 2DG to inhibit glycolysis)? Also just curious, is the control also transfected with lipofectamine and the same plasmid minus the GFP ORF?
If you want to know anymore feel free to email.
PS. Nice blog
Transient transfection vehicles can have drastic effects on cells, and it is theoretically possible that plasmid backbone DNA sequences could potentially do something. Was the control treated with the same lipofectamine conditions, but with no DNA? Or how about using the same DNA vector but with no GFP insert, as the best negative control?
Hi Beverley, to answer this question directly we are repeating the experiment with two controls, one untreated control (optiMEM only) and one lipofectamine only, which we will compare directly to the GFP plasmid. Ideally we could excise the GFP insert and include a backbone control as well (but I’m at this stage we don’t have the backbone sans GFP). I’ll post our results next week.
James
Hi James,
Love the blog, think it’s great! There should be more open science! I have heard a while back that GFP does show some toxicity in cells and I remember something about new vectors coming out with less toxic forms of GFP, but this was years ago so my memory is a bit sketchy on this. Ages ago (about 2002) we were using GFP transfected CHO cells and found we had to keep FACS sorting out the positive population (+ve for GFP) otherwise they just die out and we would get population drift towards GFP -ve. There seems to be a lot of discrepancy in the literature but a paper was published way back in 1999 (Liu et al 1999 BBRC 260(3):712-7), I haven’t had a good read of the paper so I don’t know how thorough it is, but it’s worth a look.
Anita
Hello,
It’s maybe a little bit late but,
Seahorse assay is based on two fluorescent probes (one for Oxygen and one for proton) isn’t? Are you sure the expression of GFP will not increase the background signal of the proton prob? by overlapping of the excitation/emission spectrum?
I’m planning to use some cells expressing a GFP tagged protein (easier to sort) but I’m not sure about this potential issue.