Isolation of Mitochondria From Fresh Mice Lung Tissue

Abstract

Direct analysis of isolated mitochondria enables a better understanding of lung dysfunction. Despite well-defined mitochondrial isolation protocols applicable to other tissues, such as the brain, kidney, heart, and liver, a robust and reproductive protocol has not yet been advanced for the lung. We describe a protocol for the isolation of mitochondria from lung tissue aiming for functional analyses of mitochondrial O2 consumption, transmembrane potential, reactive oxygen species (ROS) formation, ATP production, and swelling. We compared our protocol to that used for heart mitochondrial function that is well-established in the literature, and achieved similar results.

High-efficiency quantitative control of mitochondrial transfer based on droplet microfluidics and its application on muscle regeneration

“…The time spent for the transfer affects the transfer efficiency. Given the loss of mitochondrial viability after isolation ( 46 ), the transfer time was set to 2 hours. If the transfer time could be longer, then the difference in transfer efficiency among the three cases would be more evident.…”

“…We limited the mitochondrial transfer process to 2 hours, which resulted in an incomplete transfer process. If we could extend the transfer time without losing isolated mitochondria viability (46), then more isolated mitochondria could be taken up by recipient cells and the transfer efficiency could be further increased. By using such a new technology, a large number of viable C2C12 cells with mitochondrial transfer at different settings were generated for subsequent in vitro Gastrocnemius muscle tissue from all five groups was collected on day 7.…”

A potent and selective activator of large‐conductance Ca2+‐activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential

“…We understand that mitochondria do not have the same physiological parameters when isolated compared to tissue, as well as in preparations of permeabilized cells, slices of permeabilized tissues, or using a fluorophore in live tissue. However, the method for evaluating the function of isolated mitochondria is well established in the scientific literature and in our laboratory, being a good tool for evaluating conditions that affect mitochondrial function 57–59 …”

“…The mitochondrial swelling and transmembrane potential were evaluated according to previous studies, 57,59 using a spectrofluorometer (SpectraMax® M3, Molecular Devices, EUA). A mitochondrial suspension (100 μg/mL) was added to the respiration medium in the absence of respiratory substrates, at 37°C and under constant stirring.…”

Exercise Improves Redox Homeostasis and Mitochondrial Function in White Adipose Tissue

“…Immediately after euthanasia of the rats, we performed mitochondria isolation from fresh rWAT. The samples were subjected to differential centrifugation according to the modified protocol of Cladeira et al, (2021) [ 38 ] and Maciel et al, (2020) [ 39 ]. The isolation of retroperitoneal WAT mitochondria was performed according to the protocol previously published by our group [ 29 , 39 ].…”