Mitochondrial permeability transition pore assay kit

The 1 × 10⁶ cells were stained with 2 μM of JC-1 fluorescent probe (Biotium Inc., Hayward, CA, USA) for 30 min at 37 °C, centrifuged at 13,000× g for 5 min and resuspended in 0.5 mL PBS. Red fluorescence (λ excitation = 550 nm, λ emission = 600 nm), indicating polarized and undamaged mitochondria, and green fluorescence (λ excitation = 485 nm; λ emission = 535 nm), indicating depolarization and damaged mitochondria, were read using a Synergy HT Multi-Detection Microplate Reader, and expressed as relative fluorescence units (RFUs). Results were expressed as the percentage of green (depolarized)/red (polarized) mitochondria [32 (link)]. The opening of the mPTP, a second index of mitochondria depolarization and damage, was quantified in 1 × 10⁶ cells via flow cytometry (Guava EasyCyte equipped with the InCyte software v. 3.3, Millipore, Billerica, MA, USA) using the Mitochondrial Permeability Transition Pore Assay Kit (BioVision, Milpitas, CA, USA), as per manufacturer’s instructions. Autofluorescence of unstained cells was subtracted as blank. Results were expressed as the percentage of fluorescent cells over total cells.

The opening of the mPTP, considered an index of damaged mitochondria, was measured using a Mitochondrial Permeability Transition Pore Assay Kit (BioVision) according to the manufacturer’s instructions. The intracellular fluorescence was measured at an excitation λ wavelength of 488 nm on a Synergy HTX 96-well microplate reader (Bio-Tek Instruments). The results are expressed as relative fluorescence units (RFU)/mg cellular proteins.

The opening of the mPTP, a second index of mitochondria depolarization and damage, was measured with the Mitochondrial Permeability Transition Pore Assay Kit (BioVision, Milpitas, CA), as per manufacturer’s instructions, using a Guava EasyCyte flowcytometer (Millipore), equipped with the InCyte software (Millipore). 1 × 10⁵ unstained cells were used to set the threshold of autofluorescence and subtracted from the stained cells. Results were expressed as percentage of fluorescent cells over total cells.