seahorse problems

Question

Hi, we aregenerating adipocytes from human pluripotent stem cells but we face some problems with the masure of the oxygen consumption because our cells do not grow in monolayers but in multiple layers. The thing is that when we apply the classical protocol to maesure oxygen consumption with the seahorse technology in our cells, they start to deatach. has anybody faced this kind of issue? If yes do you have any suggestion? Thanks Stefania

Courtney Watts · Accepted Answer

Hi Stefania, Thank you for your question. We actually have a specific plate to deal with the very issue you are talking about. Many users working with adipocytes experience this issue. Our V28 plates were designed to help with this: https://www.agilent.com/en/products/cell-analysis/seahorse-xf-consumables/fluxpaks-microplates/seahorse-xf24-cell-culture-microplates . I also wanted to send you to our Cell Reference Database , where you can take a look at what other Seahorse users have done as far as protocols for adipocytes (and many other cells). You can change the drop downs to reflect the screenshot below, and you will see several different papers. Next, I have seen customers have more success (in addition to using the V28 plates) when extending their mixing time to 4 or 5 minutes, and shortening the wait/measure commands to 2 minutes each. I have also found some users had success when they differentiated the cells in their culture bottle and the day prior to running the cells, trypsinized the differentiated cells and plated a suitable number in the Seahorse cell plate. This to avoid too high respiration from the differentiated cells (as you are seeing). Finally, here are some links to some of our Application Notes and Technical Briefs that I think you will find useful: Measuring Bioenergetic Profiles of Human Adipocytes Measuring Functional Metabolism of Brite Adipocytes 3T3-L1 Pre-Adipocyte Differentiation

seahorse problems

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