title: Comprehensive analysis of mitochondrial permeability transition pore activity in living cells using fluorescence-imaging-based techniques.
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.
authors.affiliation: 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.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bonora
authors.affiliation: 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.
authors.forename: Claudia
authors.initials: C
authors.lastname: Morganti
authors.affiliation: 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.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Morciano
authors.affiliation: 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.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Giorgi
authors.affiliation: Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
authors.forename: Mariusz R
authors.initials: MR
authors.lastname: Wieckowski
authors.affiliation: 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.
authors.forename: Paolo
authors.identifier: 0000-0001-7108-6508
authors.initials: P
authors.lastname: Pinton
mesh_terms: D002470:Cell Survival; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D061848:Optical Imaging / Q000379:methods*
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore