Hi everyone, I'm performing the glycolysis stress test using the Seahorse XFp on primary normal human bronchial epithelial cells (NHBEs), and I'm running into some issues.
After incubating the cells in Seahorse assay media (Agilent XF DMEM + 2mM L-glutamine), no glucose/pyruvate) for 45–60 minutes at 37°C without CO₂, I notice that the cells begin to round up and detach. This seems to affect the assay — I get a good ECAR increase after glucose injection, but no significant ECAR response after oligomycin or 2-DG.
A few notes:
- I'm using rat tail collagen I for coating
- Seeding density is around 20,000 cells/well. (I have titrated cell density)
- Media is pre-warmed.
- The same assay worked well for MDCK cells, so I believe the issue is NHBE-specific.
Has anyone experience something similar with sensitive primary cells? Any tips for keeping the cells happy during the degassing step without affecting the assay?
Thanks in advance for any advice or shared experience!
I don't think it's a good idea to keep cells in the absence of glucose for over an hour. Glucose deprivation will certainly alter cellular metabolism and I modestly believe this will make subsequent measurements meaningless. I am not too surprised that your cells are detaching. It just shows how harsh is glucose deprivation.
As an alternative, I suggest keeping your cells in your standard XF medium with glucose/glutamine and add oligomycin to determine the glycolytic capacity, followed by 2DOG. Basal glycolysis should be evaluated just with the addition of 2DOG after the basal measurements.
Good luck!
I do not like this "stress protocol" and obviously never used it!
Good luck!
Best regards
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