title: Imaging H2O2 microdomains in receptor tyrosine kinases signaling.
abstract: HyPer, a ratiometric genetically encoded fluorescent sensor, is a popular tool for intracellular hydrogen peroxide detection. When expressed in cultured cells, the freely diffusing version of the sensor (HyPer-cyto) detects temporal patterns of H2O2 generation. However, rapid diffusion of the probe within the nucleocytoplasmic compartment averages the H2O2 signal even in cases of local oxidant production. Consequently, we immobilized the sensor within specific subcellular compartments allowing it to monitor local increases in H2O2. Here, we provide a protocol of ratiometric imaging and ImageJ-based quantification of H2O2 microdomains produced by cells upon physiological stimulation.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.
authors.forename: Nataliya M
authors.initials: NM
authors.lastname: Mishina
authors.forename: Kseniya N
authors.initials: KN
authors.lastname: Markvicheva
authors.forename: Arkady F
authors.initials: AF
authors.lastname: Fradkov
authors.forename: Elena V
authors.initials: EV
authors.lastname: Zagaynova
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
authors.forename: Sergey
authors.initials: S
authors.lastname: Lukyanov
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Belousov
mesh_terms: D000818:Animals; D002460:Cell Line; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide; D020794:Receptor Protein-Tyrosine Kinases