What combination of substrate I need to use on my isolated mitochondria on seahorse plate to keep them respiring?

Thanks Courtney,

I went through the recommended papers, and it helped me to understand better. What I could not find that what combination of substrate I need to use on my isolated mitochondria on seahorse plate to keep them respiring? I am using seahorse assay buffer (DMEM+Sod. Pyru+Gluc+Glutamine) for the cells to be used in the assay. 

My set-ups are:

Port-A: ADP (Different concentration from 0.5 to 4 mM)

Port-B: Oligo

Port-C: FccP

Port-D: Rot+Antimycin

I am using primary neurons from the mice to isolate the mitochondria.

Let me know!



  • Hi Guarav,

    Great, I am glad those helped! We do have a few updates that we now suggest. These will be released at some point in written form, but please see below for the time being.  Regarding the solution that you should be using during the assay: this will be 1X MAS - see screenshot below. This assay does not use Seahorse media at all as iso mito rather than intact cells are being used.  

    • When making 3X stock MAS: omit the BSA
    • When making the drug injection solutions, omit BSA
    • Substrates may also be omitted from the drug injection solutions if using multiple substrates (this is for convenience)
    • Do ensure that BSA is added to MAS for mitochondria adhesion and bulk media added after the adhesion step.
    • Ensure that both ADP and an oxidizable substrate are present for the adherence step.
    • At least 2 measurements are now suggested after each injection
    • It is encouraged to begin the assay in State 3 respiration, i.e. in the presence of ADP

    Regarding the combination of substrates/inhibitors needed, this will depend on which complexes you are interested in testing and/or what question(s) you are trying to answer with your research. I think the below chart from this paper will be helpful.  This paper relates to PMP, but there is some overlap between PMP and iso mito.

    I also want to make sure you are aware that the Agilent Mito Stress Test is not applicable to iso mito either as this assay kit was designed for intact cells, and they concentrations in the kit's reagents will not be sufficient for iso mito.

    Thank you and please let me know if this helps or you have any other questions!


  • Dear Courtney,

    Thank you for your reply. I read the paper you attached to answer my question. But I was not aware that Mito Stress kit was not compatible with Isolated mitochondria and ofcourse my experiment did not work. 

    I am isolating mitochondria from neuron cells and I want to see whether isolated mitochondria are metabolically active and respiration competent. I will be using this mitochondria for transplantation later on with initially co-culture and later on in in-vivo. Below is the link of the protocol we used from Boston hospital, USA and it's a rapid protocol to isolate mitochondria.

    Could you suggest me what experiment I should opt for on Seahorse to analyze my isolated mitochondria?

    I look forward to hearing from you.

    Best Regards,


  • Hi Gyarav,

    You are very welcome. The assays we have in this paper are Coupling or Electron Flow.  The Coupling Assay examines the degree of coupling between the electron transport chain (ETC), and the oxidative phosphorylation machinery (OXPHOS), and can distinguish between ETC and OXPHOS with respect to mitochondrial function/dysfunction. The Electron Flow assay examines sequential electron flow through different complexes of the electron transport chain, which can identify the mechanism of mitochondrial dysfunction or modulation.  The assay choice will be up to your discretion. 

    Please let me know if this helps.

    Thank you,


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