Hi, guys, recently I am performing the glycolysis seahorse assay in endothelial cells. I followed the protocol as suggested, but the ECAR didn't increase after adding Oligomycin. Do you think the oligomycin concentration is too high or the PH value is so low that affects the ECAR value? In the meantime, I also noticed other publications, they got the same data, the cell didn't respond to the oligomycin. Anyone who has suggestions on this experiment is appreciated.
Dear Liu Bin
Based on the plot you have shared I noticed that your basal reading is quite low (the first reading is in negative values and then it spikes upwards). Do you have any ideas why this happened?
As the first steps, I would recommend to confirm that the cell density is appropriate (meaning they should be neither over confluent nor should there be any empty spaces)
also have you checked the pH of the media being used for the assay? I normally prepare the medium fresh and use it up within 3-4 hours.
in general it is always a good idea to perform a dose dependent titration curve for figuring out the best oligomycin concentration.
(I also prepare the oligomycin solution fresh in the assay medium where the pH has been set to 7.4)
Hope this will help to improve your results.
Hi, Shambhabi Chatterjee, thanks for your answer. Your suggestions are very helpful. As you mentioned, the PH value and the cell density are very important, and we already figured out and optimized our protocol. Right now the basal reading is normal, I think it is due to the right PH value we have adjusted. Moreover, all the mediums that we used were fresh, while the dose-dependent titration curve for figuring out the best oligomycin concentration is my next top priority. Another question is: the assay medium we used is DMEM +2 uM glutamine, I am not sure it is suitable for endothelial cells. Because the suggested protocol I used from the Agilent company is for cancer cells. And I searched the reference, I didn't find more details about which medium they used for assay medium.
Dear Liu Bin
Thank you for the detailed answer.
Personally I have not performed seahorse assays for endothelial cells (ECs) so far, but I could refer this article which also discusses the role of glutamine for ECs. The authors have used DMEM+ 2mM Glutamine. maybe you can have a look at their methods section for more details- Article Endothelial tip cells in vitro are less glycolytic and have ...
For the assay medium pls try to use medium without Phenol red. I have observed much more consistent results when the assay medium does not contain phenol red.
Another factor that causes weird trends in the final data are 'bubbles' that occur during experiment run. To make sure the wells did not have such an issue you can look at the O2 curve from the Wave analysis software. If the O2 curve is not smooth then most likely bubbles were introduced somehow while mixing or injecting and these wells then need to be eliminated from the final analysis.
Hope this will help in troubleshooting!
Hi, Shambhabi Chatterjee, thanks for your detailed answer. I read the paper you linked and it is the same paper that I referred to(same cell numbers, compounds concentration, and medium). The key things you mentioned are that the O2 curve, I will check it. Thanks again, I will run the assay again this Friday. And we will see how it is going.
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