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Isolation of mitochondria is a crucial method for examining molecular details of this organelle's manifold functions. Historically, mitochondrial isolations required large amounts of sample material which impeded their isolation from cultured cells. We have therefore developed a method allowing for controlled and reproducible isolation of intact and functional mitochondria from diverse cell types in culture. Here we provide a methodological update of this approach together with a protocol for the subsequent analysis of such isolated mitochondria by electron microscopy. Combining the isolation procedure with this powerful imaging method can reveal ultrastructural mitochondrial peculiarities in disease settings that might not be evident in intact cells and allows for assessment of mitochondrial membrane integrity and sample purity.

Mitochondria are key organelles for cellular energy production and cell death decisions. Consequently, a plethora of conditions which are toxic to cells are known to directly attack these organelles. However, mitochondria originating from different tissues differ in their sensitivity to toxic insults. Thus, in order to predict the potential organ-specific toxicity of a given drug or pathological condition at the mitochondrial level, test settings are needed that directly compare the responses and vulnerabilities of mitochondria from different organs. As a prerequisite for such test strategies, we provide here a robust, prompt, and easy-to-follow step-by-step protocol to simultaneously isolate functional and intact mitochondria from rat liver, kidney, heart, and brain. This isolation procedure ensures mitochondrial preparations of comparable purity and reproducible quantities which can be subsequently analyzed for organ-specific mitochondrial toxicity.

We recently reported a new method to isolate functionally intact mitochondria from cell culture and small tissue samples (Schmitt et al., Anal Biochem 443(1):66-74, 2013). This method comprises a semi-automated cell rupture, termed pump controlled cell rupture system (PCC), which can be precisely adjusted to the specific cellular source of isolation and which can be tightly controlled (Schmitt et al., Anal Biochem 443(1):66-74, 2013). Here we provide a detailed hands-on protocol of this PCC method which results in an efficient cell breakage but preserving the mitochondrial integrity. Upon subsequent purification steps, the obtained mitochondrial fraction meets the quality and purity required for molecular analyses, e.g. proteomic comparisons, as well as for biochemical analyses, e.g. determination of diverse enzymatic activities.