Talk:MiPNet17.02 PBI-Shredder manual

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Advantages and disadvantages

Advantages

  • Standardized tissue preparations for obtaining disrupted cells with functional mitochondria.
  • Highly reproducible results.
  • Easy handling, especially for beginners.
  • Minimum processing time of 10 min.
  • Closed Shredder-Tubes ensure safety throughout the entire sample preparation process.
  • Either the homogenate may be used directly for HRR, or the homogenization process may be followed by further isolation of mitochondria.
  • The homogenate is suitable for optical measurements (e.g. O2k-Fluorometry with safranin for detection of mt-membrane potential) where a homogenous suspension is required.

Disadvantages

  • A fraction of mitochondria is lost (c. 50% in our preparations), therefore about twice the amount of tissue is required compared to permeabilized fibres or homogenization of the total tissue with a potter.
  • Since not all mitochondria are obtained from the tissue, tissue mass-specific mitochondrial respiratory capacity can be measured only on the basis of additional measurements of a mitochondrial marker (e.g. CS activity) in the total tissue and in the homogenate, to quantify the mt-yield and refer respiration of the homogenate to Ww of tissue.
  • Since not all mitochondria are obtained from the tissue, it is difficult to evaluate if specific mitochondrial types are enriched or a representative subsample of all mitochondria is obtained.
  • The cytochrome c effect is larger in homogenate compared to permeabilized fibres, hence a small degree of functional impairment of mitochondria is caused by the homogenization process.


Shredder vs Dounce

  • Question: Can PBI-Shredder completely replace the traditional Dounce Potter-Elvehjem for tissue homogenate preparation?
  • Answer: No, the Shredder does not replace the traditional Dounce homogenizer (or Potter-Elvehjem). The Dounce homogenizer is generally used in the course of preparing isolated mitochondria, where homogenization is followed by differential centrifugation. The Shredder is supposed to be used for homogenization only, i.e. the homogenate is directly used in respirometric measurements. This is advised in cases where it is either important to minimize the time required for sample preparation or where a loss of sample due to differential centrifugation has to be avoided. It must be kept in mind that the use of the Shredder must be optimized for each specific application, i.e. time and force applied during homogenization need to be tested. Further, it needs some practice before good quality samples can be obtained. However, if preliminary studies are thoroughly conducted we found that good quality samples can be prepared in relatively little time and with very small samples.
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