Simultaneous Acquisition of Mitochondrial Calcium Retention Capacity and Swelling to Measure Permeability Transition Sensitivity

Abstract

The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca²⁺). Respiring mitochondria will intake available Ca²⁺ into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca²⁺ taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca²⁺ retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca²⁺ until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca²⁺-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca²⁺ added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.