We recently purchased a Seahorse Analyzer and are interested in other scientists' experience with the different the different kits that are available.
in principle yes, however, some times pH change are neglected so will be the result. Which I have seen in my exp. Absolute is pH of the measuring medium at least for mitostress and rest of the components can be used from different origin.
Why would you like to use the kit when you can buy the individual components at much lower price and be sure of their purity and quality
Thanks for your input, Sandipan. We don't have experience purchasing kits or individual components - we are currently following the recommendation of our service rep. We will definitely look into individual components as per your advice.
The Glycolysis stress kit contains Glucose, Oligomycin and 2-deoxy-D-glucose. You can buy them from Sigma and prepare individual stock solution.
The mito test kit contains Rotenone, Antimycin, Oligomycin and FCCP all of these can be bought from Sigma and are pretty stable under their recommeded storage conditions.
Ask you rep why you should buy this kit instead of buying individual components and see what he/she says
Thanks again Sandipan. We have all of these reagents in the lab... I look forward to my next conversation with the service rep.
-Nate
Cool I thought so. Any mitochondria lab will have these basic mito reagents :)
I don't think the SeaHorse kits are supposed to keep the pH constant. At least none of their components suggest it unless the solutions are prepared in buffers. So I think you can measure pH changes after Oligomycin addition.
Jonas/Sandipan I am looking to buy the individual components right now! The Mito stress and Glycolysis kits are SO EXPENSIVE! Did have any luck? The main thing that makes me nervous is the Oligomycin being a combination of A, B and C. Although you can order the mix from Sigma I am just wondering whether it will have the exact MW on the bottle and whether I will be able to accurately make a specific molarity. Also - whether there will be differences in the ratio of the three oligos and if this will make a difference.
Also - The spec sheet for the oligo A/B/C mix states to dissolve in DMSO or ethanol. Whereas the kit resuspends in seahorse media. I just get worried that maybe I won't be able to dissolve the sigma Oligo in media.
Any thoughts??
Hi everyone, I'm a new PhD student working on DC metabolism and gonna use seahorse machine for the first time. I'm the first in my lab to use the machine so don't have much guidance. From the above responses, I understand that rather than using the kit, the experiments should work fine with purchasing individual components. I'm wondering how about the media? Do you need to purchase the seahorse media or can we use the normal RPMI? Thanks.
Hey Alice,
The media is really important because of the buffering capacity. You need to be able to see the differences in pH without the interference of buffers. So seahorse media is most likely required.
Also - just be careful of what you resuspend the individual components in. I had to test out the effect of DMSO (which had no effect) but worth making sure it doesn't make any changes.
Good luck!
Helen Lenthall Did you have any luck getting specific catalog numbers for reagents? I'm going through the same thing now looking at options between Cayman and Millipore Sigma, and I'm noticing 1) a wide difference in price and 2) differences in some of the technical details, even for apparently comparable mixes of oligomycin complexes. I'd appreciate any advice you might have!
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