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

Question

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! 
 
 Best, 
 
 Gaurav

Courtney Watts · Accepted Answer

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! Courtney

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

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