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.