Hi,
I am trying to establish a protocol for heart mitochondria evaluation in the Oxytherm+System (Hansatech Ltd). Should I add rotenone or not? I was using succinate+rotenone at the beginning of the record, but a person told me that rotenone sticks in the chamber and accumulate there. Is it true? Because almost all the papers that I saw used rotenone.
Thank you
Hello, yes rotenone can accumulate in the chamber as well as other compounds as well. You may need to use a yeast solution to add to the chamber before beginning experiments. Yeasts clean up accumulating debris.
Thank you for your answer, Welligton. Do you know how can I buy? How the protocol works? I am new in this field, so I do not know where to start
My experience is, that not just rotenone sticks to the membrane and the wall of the chamber but uncouplers like dinitrophenol or F/CCCP too. I rinse with methanol/ethanol followed by H2O between experiments. I also change the membrane after using "sticky" drugs/chemicals.
Good luck!
Thank you, Gisela Beutner ! We are doing the same way.
I saw that you are an expert in rat heart mitochondria.
Could you please help me with another things? I am using 200 ug/mL of mitos and doing the curves in the following sequence: 5.5 mM succinate, 2uM rotenone, 500 uM ADP, 2 uM Oligomycin and 1uM FCCP .
However, I saw other papers using succinate and rotenone after FCCP. Do you think that we are doing right?
And also I am not having like a plateau when I put oligomycin, is it normal?
Dear Nobr Ribeiro,
please go to my page “Alexander Panov” on Research Gate and find my recent papers on heart mitochondria metabolism. Also. You can Also find my book Practical mitochondriology. The paper version you can by on Amazon just for 5 bucks. Do not use the old a methodology. It is absolete. You can wash away rotenone with thick susp3nsion of liver mitochondria. But basically, you do not need to use rotenone at all.
Hello Carolina,
to answer your question whether to add succinate and rotenone after FCCP? I would not do it, because if the mitochondria are already uncoupled and presumably at an oxygen consumption rate comarable ,to Vmax I would not expect to see a response .
Gisela
Dear colleague, using succinct+rotenone with heart mitochondria brings no useful information to understand hearts’ physiology or pathology. On my page in Research gate you can find my recent papers published in EC Cardiology. For the RHM physiological substrates mixtures must include palmitoyl-carnitine (0.02 mM in 50% ethanol) plus any substrates: succinate, glutamate, pyruvate or their combinations.
Thank you Alexander V Panov , I have read your book and it is helping a lot! It is a very nice knowledge sharing.
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