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Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques

Nature Protocols volume 11, pages 1067–1080 (2016)Cite this article

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Abstract

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein–cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take ∼1 d for methods (i) and (ii), and ∼3 d for method (iii). The entire experiment, including analyses, takes ∼2 h.

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Figure 1: Representative images and kinetics of HeLa cells stained using the Co2+–calcein technique.
Figure 2: Representative images and kinetics of HeLa cells stained with TMRM (Fire LUT was applied).
Figure 3: Representative images and kinetics of HeLa cells expressing mitochondrially targeted GFP.

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Acknowledgements

P.P. is grateful to Camilla degli Scrovegni for continuous support. This research was supported by the Italian Ministry of Education, University and Research (COFIN no. 20129JLHSY_002, FIRB no. RBAP11FXBC_002, and Futuro in Ricerca no. RBFR10EGVP_001), local funds from the University of Ferrara and the Italian Ministry of Health to P.P. and C.G., Telethon (GGP15219/B), the Italian Association for Cancer Research (IG-14442 and MFAG-13521 to P.P. and C.G.), and the Italian Cystic Fibrosis Research Foundation (19/2014) to P.P. M.R.W. was supported by the National Science Centre, Poland (grant 2014/15/B/NZ1/00490), grant W100/HFSC/2011, and HFSP grant RGP0027/2011.

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  1. Massimo Bonora, Claudia Morganti and Giampaolo Morciano: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy

    Massimo Bonora, Claudia Morganti, Giampaolo Morciano, Carlotta Giorgi & Paolo Pinton

  2. Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland

    Mariusz R Wieckowski

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Contributions

M.B., C.M., G.M., C.G., M.R.W., and P.P. contributed extensively to the writing of this paper. M.B., G.M., and C.M. performed the experiments, analyzed data, and generated visual guides.

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Correspondence to Paolo Pinton.

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Integrated supplementary information

Supplementary Figure 1 Typical improper results

Example of typical experimental artifacts in the Co2+-calcein (A), mitochondrial membrane potential (B) and mitochondrial morphology assays (C). For solutions to these problem, refer to Table 2.

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Bonora, M., Morganti, C., Morciano, G. et al. Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques. Nat Protoc 11, 1067–1080 (2016). https://doi.org/10.1038/nprot.2016.064

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