Important methodological aspects that should be taken into account during the research of isolated mitochondria

Isolated mitochondria have been widely used for the study of energetic functioning of these important cellular organelles in a physiological or pathophysiological state. This is due, on the one part, to the fact that isolated mitochondria are relatively easy to isolate from a great variety of animal tissues, and on the other part to the fact that their energetic functioning is relatively easy to study experimentally. Nevertheless, they have a great disadvantage because of the fact that these biological structures can easily undergo structural-functional changes during their in vitro handling, causing many conclusions reported in the research of isolated mitochondria to be merely the fruit of experimental artifacts. The present review describes a series of important methodological aspects that should be taken into account in order to obtain reliable results in the study of the energetic functioning (and of other aspects) of isolated mitochondria. •The tissue should be sliced rapidly; the use of scissors can take up a lot of time; the use of Fig. 1 press is recommended.•To avoid mitochondrial aggregates not use high concentrations of Mg2+ and/or cytochrome c in a low ionic strength buffer.•Many other factors favor the formation of mitochondrial clumps, implicating artefactual mitochondrial observations.•It is fundamental to test if mitochondrial clustering is taking place in ongoing tests.•It is discussed mitochondrial literature, in which its conclusions are based on artefactual experimental observations.