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This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.
Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.
Several important mitochondrial enzymes and signaling molecules are subject to thiol redox regulation1. Moreover, mitochondria are a major cellular source of reactive oxygen species and are selectively vulnerable to oxidative damage2. Accordingly, the mitochondrial redox potential directly affects bioenergetics, cell signaling, mitochondrial function, and ultimately cell viability3,4. The mitochondrial matrix contains high amounts (1-15 mM) of the thiol-disulfide redox buffer glutathione (GSH) to maintain redox homeostasis and mount antioxidant defenses
All animal experiments conformed to national and institutional guidelines, including the Council Directive 2010/63/EU of the European Parliament, and had full Home Office ethical approval (University of Heidelberg Animal Welfare Office and Regierungspraesidium Karlsruhe, licenses T14/21 and T13/21). Primary hippocampal and cortical neurons were prepared from newborn mouse or rat pups according to standard procedures and were maintained for 12-14 days as previously described13.
1. Preparation of solutions
Quantification of differences in steady-state mitochondrial redox state after growth factor withdrawal
To demonstrate the quantification of steady-state differences in mitochondrial redox state, primary neurons grown in standard medium were compared to neurons cultured without growth factors for 48 h before imaging. Growth factor withdrawal results in apoptotic neuronal cell death after 72 h16. Cells were imaged after 48 h to test if this is preceded by changes in mitochondr.......
Quantitative and dynamic measurements of the mitochondrial redox state provide important information about mitochondrial and cellular physiology. Several fluorogenic chemical probes are available that detect reactive oxygen species, "redox stress," or "oxidative stress." However, the latter terms are not well-defined and often lack specificity9,17,18. Compared to chemical dyes, Grx1-roGFP2 offers several advantag.......
The authors declare that they have no conflict of interest.
This work was supported by the Deutsche Forschungsgemeinschaft (BA 3679/5-1; FOR 2289: BA 3679/4-2). A.K. is supported by an ERASMUS+ fellowship. We thank Iris Bünzli-Ehret, Rita Rosner, and Andrea Schlicksupp for the preparation of primary neurons. We thank Dr. Tobias Dick for providing pLPCX-mito-Grx1-roGFP2. Experiments shown in Figure 4 were performed at the Nikon Imaging Center, University of Heidelberg. Figure 2 was prepared with BioRender.com.
....Name | Company | Catalog Number | Comments |
reagents | |||
Calcium chloride (CaCl2·2H2O) | Sigma-Aldrich | C3306 | |
Diamide (DA) | Sigma-Aldrich | D3648 | |
Dithiothreitol (DTT) | Carl Roth GmbH | 6908.1 | |
Glucose (2.5 M stock solution) | Sigma-Aldrich | G8769 | |
Glucose | Sigma-Aldrich | G7528 | |
Glycine | neoFroxx GmbH | LC-4522.2 | |
HEPES (1 M stock solution) | Sigma-Aldrich | 15630-080 | |
HEPES | Sigma-Aldrich | H4034 | |
Magnesium chloride (MgCl2·6H2O) | Sigma-Aldrich | 442611-M | |
N-methyl-D-aspartate (NMDA) | Sigma-Aldrich | M3262 | |
Potassium chloride (KCl) | Sigma-Aldrich | P3911 | |
Sodium chloride (NaCl) | neoFroxx GmbH | LC-5932.1 | |
Sodium pyruvate (0.1 M stock solution) | Sigma-Aldrich | S8636 | |
Sodium pyruvate | Sigma-Aldrich | P8574 | |
Sucrose | Carl Roth GmbH | 4621.1 | |
Tetramethylrhodamine ethyl ester perchlorate (TMRE) | Sigma-Aldrich | 87917 | |
equipment | |||
imaging chamber | Life Imaging Services (Basel, Switzerland) | 10920 | Ludin Chamber Type 3 for Ø12mm coverslips |
laser scanning confocal microscope, microscope | Leica | DMI6000 | |
laser scanning confocal microscope, scanning unit | Leica | SP8 | |
peristaltic pump | VWR | PP1080 181-4001 | |
spinning disc confocal microscope, camera | Hamamatsu | C9100-02 EMCCD | |
spinning disc confocal microscope, incubationsystem | TokaiHit | INU-ZILCF-F1 | |
spinning disc confocal microscope, microscope | Nikon | Ti microscope | |
spinning disc confocal microscope, scanning unit | Yokagawa | CSU-X1 | |
software | |||
FIJI | https://fiji.sc | ||
StackReg plugin | https://github.com/fiji-BIG/StackReg/blob/master/src/main/java/StackReg_.java | ||
TurboReg plugin | https://github.com/fiji-BIG/TurboReg/blob/master/src/main/java/TurboReg_.java |
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