Plate-Based Assays for the Characterization of Mitochondrial and Cellular Phenotypes.
Methods Mol Biol
; 2746: 1-20, 2024.
Article
in En
| MEDLINE
| ID: mdl-38070076
ABSTRACT
The mitochondria are essential to eukaryotic life, acting as key drivers of energy generation while also being involved in the regulation of many cellular processes including apoptosis, cell proliferation, calcium homeostasis, and metabolism. Mitochondrial diseases which disrupt these processes lead to a diverse range of pathologies and lack consistency in symptom presentation. In disease, mitochondrial activity and energy homeostasis can be adapted to cellular requirements, and studies using Dictyostelium and human lymphoblastoid cell lines have shown that such changes can be facilitated by the key cellular and energy regulators, TORC1 and AMPK. Fluorescence-based assays are increasingly utilized to measure mitochondrial and cell signalling function in mitochondrial disease research. Here, we describe a streamlined method for the simultaneous measurement of mitochondrial mass, membrane potential, and reactive oxygen species production using MitoTracker Green™ FM, MitoTracker Red™ CMXRos, and DCFH-DA probes. This protocol has been adapted for both Dictyostelium and human lymphoblastoid cell lines. We also describe a method for assessing TORC1 and AMPK activity simultaneously in lymphoblastoid cells. These techniques allow for the characterization of mitochondrial defects in a rapid and easy to implement manner.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Mitochondrial Diseases
/
Dictyostelium
Limits:
Humans
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2024
Document type:
Article
Affiliation country:
Australia
Country of publication:
United States