Monitoring Nicotinamide Adenine Dinucleotide and its phosphorylated redox metabolism using genetically encoded fluorescent biosensors

Apollo-NADP + reveals in vivo adaptation of NADPH/NADP + metabolism in electrically activated pancreatic β cells

“…Wide-field fluorescence imaging of 5-dpf zebrafish embryos was performed on an Axio Zoom V16 (Zeiss). Two-photon fluorescence anisotropy imaging was performed on an LSM710 microscope (Zeiss) as previously described ( 7 ). Cell lines were imaged using a 63×/1.4 numerical aperture (NA) oil immersion objective, and zebrafish pancreatic islets were imaged using a 40×/1.3 NA oil immersion objective.…”

“…Steady-state fluorescence anisotropy images were analyzed as previously described ( 7 ). Briefly, custom ImageJ and Python scripts were used to subtract the background, apply a threshold to the upper and lower fifth percentile of the range of intensity values, calculate the anisotropy value for every pixel including correcting for high NA skewing of the anisotropy, and report the average anisotropy of regions of interest (ROIs).…”

“…Despite the importance of NADPH/NADP + redox homeostasis to cell survival, redox dynamics are understudied due to limitations in methodology. Several genetically encoded sensors were developed recently to address these limitations ( 7 ). Our group developed Apollo-NADP + , a genetically encoded sensor that measures NADPH/NADP + redox changes as reflected in the steady-state fluorescence anisotropy due to homologous fluorescence resonance energy transfer (homo-FRET) ( 8 ).…”

Measuring ROS and redox markers in plant cells

“…Fluorescent reporter proteins can be specific to the oxidised and reduced forms of NAD(P)H and can provide additional subcellular specificity through targeting strategies. Many different NAD(P)(H) sensors exist (reviewed by Kyere-Yeboah et al 154 ) but only SoNar, 155 iNap 156 and Peredox 157 have been expressed to date in plants. 158,159,203 All three sensor proteins are based on the bacterial Rex NADH binding protein from Thermus aquaticus fused to either cpYFP or cpT-Sapphire, with SoNar and Peredox sensitive to the NADH : NAD + ratio 155,157 whereas iNap detects NADPH alone.…”