Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.
Methods in molecular biology (Clifton, N.J.)
confidence
Key findings
Protocols for monitoring intraperoxisomal NADPH levels and NAD+/NADH redox state using fluorescent protein sensors (iNAP1 and SoNar); no clinical/biological endpoints reported.
View source on PubMed (PMID 36952186) ↗
- Sample size
- Not reported
- Population
- Living cells (in vitro)
- Dosing
- Not reported
- Duration
- Not reported
- Route
- Not reported
- Blinding
- not_reported
- Controls
- none
- Drug class
- coenzyme
Full abstract
The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.