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Talk:Tissue homogenate

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MiPNet discussion forum: tissue homogenate (2015-11-10)

Bjørg Egelandsdal

We are working with Shredder permeabilized fibres from salmon muscle. We have done this before, but now suddenly we only get 1/3 of the OCR that we used to. What are the most common mistakes? We have run out of ideas, thanking you in advance.

Teresa Ea

Hi Bjorg,
I have noticed a few things while working with brain tissue homogenate that may help you:
  1. Tissue preparation: It's important that this technique is gentle and consistent. Preparations that are too rigorous on the tissue can end up damaging or killing the mitochondria all together. It's also a good idea to have the same person prepare the tissue every time, just to keep things consistent.
  2. Time between preparation and experiment: In my experience the sooner the tissue is placed into the chamber, the better. Sitting on ice for even an additional hour before starting the experiment can lead to reduced mitochondrial functioning.
  3. Chamber contamination: This is usually the main problem, and can easily be avoiding by thoroughly cleaning the chambers after every experiment. Residual tissue can cling to the stir bar, so make sure to remove the stir bar from the chamber and clean it after every experiment. There could also be residual proteins or inhibitors/uncouplers that cling to the chamber. The OROBOROS lab has a great cleaning SOP on the website that goes into more detail about cleaning and potential contamination.

MiPNet discussion forum: use of protease inhibitors for tissue homogenate (2016-04-12)

Marko Ljubkovic

Has anyone tried to add some protease inhibitor to the cardiac (tissue) homogenates prepared for measurements of HRR and other parameters detected with LED2 sensor (like Amplex Red). Thinking about the preparation procedure, it seems to me that the quality of the prep could be somewhat degraded by the proteases liberated during the homogenization with the Shredder. Do you have any data or thoughts on this?

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