Isolation of mitochondria with well preserved function is a prerequisite for the measurement of mitochondrial respiration. In the literature a wide diversity of buffers with different composition for sample homogenization can be found as well as various settings as related to centrifugation force and time for separation of mitochondria. The appropriate method has to be selected to meet specific needs for purity and functionality of isolated mitochondria, additionally taking into account time and cost requirements of the procedure. Functional mitochondria can be prepared only from fresh and not from frozen tissue. It is pivotal that during the entire isolation procedure the samples and buffers are kept on ice.
Mitochondria from brain and liver can be separated by simple differential centrifugation of tissue homogenate. Bovine serum albumin should be used to bind free fatty acids released from membranes during the isolation procedure, as these may act as uncouplers. For isolation of mitochondria from heart and skeletal muscle, a protease should be added in the homogenization step so as to release mitochondria from the interfibrillar space.
The listed MiPNet procedures for isolation of mitochondria from rat tissues (brain, liver and heart) were evaluated in the Pharmacobiochemical Laboratory, 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovak Republic as optimal for a good yield of mitochondria with satisfactory purity and well preserved function for measurements of mitochondrial respiration.
|MiPNet21.17 BloodCellsIsolation||»Versions||Intact cells|
|MiPNet20.08 IsolationRatLiver-mt||»Versions||Isolated mitochondria||Liver||Rat||2016-11-18|
|MiPNet20.07 IsolationRatBrain-mt||»Versions||Isolated mitochondria||Nervous system||Rat||2016-06-18|
|MiPNet20.15 IsolationRatHeart-mt||»Versions||Isolated mitochondria||Heart||Rat||2016-03-25|
|MiPNet14.14 PermeabilizedFiberPreparation||»Versions||Permeabilized tissue||Heart|
|MiPNet20.06 IsolationMouseHeart-mt||»Versions||Isolated mitochondria||Heart||Mouse||2015-06-22|
|MiPNet17.02 PBI-Shredder manual||» Versions||Homogenate||2015-06-19|
|MiPNet17.03 Shredder vs Fibers||»Versions||Homogenate||Heart|
|MiPNet20.09 IsolationBeefHeart-mt||»Versions||Isolated mitochondria||Heart||Bovines||2015-06-15|
|MiPNet11.05 Mitos-PermeabilizedCells||»Versions||Permeabilized cells|
|MiPNet08.13 MitoIsolation-RLM||»Versions||Isolated mitochondria||Liver||Rat||2010-08-03|
|MiPNet17.02 PBI-Shredder manual||» Versions||2015-06-19|
|O2k-Procedures » Link|
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|» O2k-Chemicals and media|
|» O2k-Mitochondrial preparations|
|» O2k-Procedures: Mitochondrial and marker-enzymes|