{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:27:35Z","timestamp":1787740055472,"version":"build-2784847793"},"reference-count":28,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":13,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["24\u201074\u201010106"],"award-info":[{"award-number":["24\u201074\u201010106"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,8]]},"abstract":"Abstract<\/jats:title>\n \n Hydrogen peroxide (H\n 2<\/jats:sub>\n O\n 2<\/jats:sub>\n ) plays an important role in liver metabolism as a second messenger under physiological conditions, as well as in the development of oxidative stress and pathology. The major metabolic functions of the liver are carried out by hepatocytes, the parenchymal cells that constitute most of the liver mass. The study of oxidative stress in the liver, in particular the determination of a causal relationship between pathological progression and H\n 2<\/jats:sub>\n O\n 2<\/jats:sub>\n production, requires an optimal model system. The chemogenetic system based on yeast\n d<\/jats:sc>\n \u2010amino acid oxidase (DAAO) fused to the C\u2010terminus of the HyPer7 biosensor has been established as a tool for controlled H\n 2<\/jats:sub>\n O\n 2<\/jats:sub>\n production and examination of intracellular H\n 2<\/jats:sub>\n O\n 2<\/jats:sub>\n dynamics in vitro and in vivo. This article details adeno associated virus (AAV)\u2013mediated targeted delivery of the HyPer7\u2010DAAO transgene into mouse liver, followed by isolation of primary hepatocytes for quantitative evaluation of intracellular H\n 2<\/jats:sub>\n O\n 2<\/jats:sub>\n dynamics based on ratiometric HyPer7 fluorescence acquired by wide\u2010field microscopy and subsequent image analysis. \u00a9 2026 Wiley Periodicals LLC.\n <\/jats:p>\n \n Basic Protocol 1<\/jats:bold>\n : AAVs production and purification\n <\/jats:p>\n \n Basic Protocol 2<\/jats:bold>\n : Intraparenchymal injection of AAVs into mouse liver\n <\/jats:p>\n \n Basic Protocol 3<\/jats:bold>\n : Primary mouse hepatocyte culture preparation\n <\/jats:p>\n \n Basic Protocol 4<\/jats:bold>\n : Wide\u2010field fluorescence microscopy of HyPer7 in primary mouse hepatocyte culture\n <\/jats:p>\n \n Basic Protocol 5<\/jats:bold>\n : Time series processing\n <\/jats:p>","DOI":"10.1002\/cpz1.70430","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T12:56:35Z","timestamp":1786712195000},"source":"Crossref","is-referenced-by-count":0,"title":["Hydrogen Peroxide Imaging via HyPer7 Probe in Adeno Associated Virus\u2013Transduced Primary Mouse Hepatocytes"],"prefix":"10.1002","volume":"6","author":[{"given":"Varvara D.","family":"Starodubova","sequence":"first","affiliation":[{"name":"Pirogov Russian National Research Medical University Moscow Russia"},{"name":"Shemyakin\u2010Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Science Moscow Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander A.","family":"Moshchenko","sequence":"additional","affiliation":[{"name":"Center for Genetic Reprogramming and Gene Therapy, Federal Center of Brain Research and Neurotechnologies Federal Medical Biological Agency Moscow Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dmitry V.","family":"Teplyakov","sequence":"additional","affiliation":[{"name":"Center for Genetic Reprogramming and Gene Therapy, Federal Center of Brain Research and Neurotechnologies Federal Medical Biological Agency Moscow Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arina G.","family":"Shokhina","sequence":"additional","affiliation":[{"name":"Pirogov Russian National Research Medical University Moscow Russia"},{"name":"Center for Genetic Reprogramming and Gene Therapy, Federal Center of Brain Research and Neurotechnologies Federal Medical Biological Agency Moscow Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"e_1_2_11_2_1_1","doi-asserted-by":"publisher","DOI":"10.5604\/16652681.1193701"},{"key":"e_1_2_11_2_2_1","doi-asserted-by":"publisher","DOI":"10.1089\/hgtb.2011.034"},{"key":"e_1_2_11_2_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth866"},{"key":"e_1_2_11_2_4_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.43.3.506"},{"key":"e_1_2_11_2_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.757"},{"key":"e_1_2_11_2_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpch.20"},{"key":"e_1_2_11_2_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596\u2010018\u20100097\u20103"},{"key":"e_1_2_11_2_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2020.100086"},{"key":"e_1_2_11_2_9_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpmo.56"},{"key":"e_1_2_11_2_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2018.05.001"},{"key":"e_1_2_11_2_11_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142905.hga03cs00"},{"key":"e_1_2_11_2_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2012.04.004"},{"key":"e_1_2_11_2_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.snb.2020.128054"},{"key":"e_1_2_11_2_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.redox.2023.102604"},{"key":"e_1_2_11_2_15_1","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2013.5618"},{"key":"e_1_2_11_2_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/cphy.c200021"},{"key":"e_1_2_11_2_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0016\u20105085(98)70643\u20102"},{"key":"e_1_2_11_2_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2020.02.003"},{"key":"e_1_2_11_2_19_1","doi-asserted-by":"publisher","DOI":"10.3390\/livers1010002"},{"key":"e_1_2_11_2_20_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R113.544635"},{"key":"e_1_2_11_2_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010018\u201006533\u20102"},{"key":"e_1_2_11_2_22_1","doi-asserted-by":"publisher","DOI":"10.1134\/S0006297925602473"},{"key":"e_1_2_11_2_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010023\u201038961\u20100"},{"key":"e_1_2_11_2_24_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3300938"},{"key":"e_1_2_11_3_2_1","unstructured":"Pak et\u00a0al. (2020). As above."},{"key":"e_1_2_11_3_3_1","unstructured":"Describes the development and characterization of HyPer7 the genetically encoded H2O2biosensor used in this article. The authors demonstrate key improvements over earlier HyPer variants: increased brightness a wider dynamic range and critically pH insensitivity (a major limitation of previous\u2010generation probes). The article provides spectroscopic characterization of HyPer7 (excitation maxima at 400 and 499 nm emission at 516 nm) validation of its selectivity for H2O2over other ROS and biological applications including imaging of H2O2gradients in migrating cells and mitochondrial H2O2topology. These properties make HyPer7 the sensor of choice for the ratiometric imaging protocols described here."},{"key":"e_1_2_11_3_4_1","unstructured":"Bogdanova et\u00a0al. (2017). As above."},{"key":"e_1_2_11_3_5_1","unstructured":"Describes the original DAAO\/HyPer\u2010based chemogenetic system for spatiotemporally controlled intracellular H2O2production. The protocol employs yeastd\u2010amino acid oxidase (DAAO from Rhodotorula gracilis) fused or co\u2010expressed with HyPer biosensor variants (HyPer2 and HyPerRed) to enable inducible substrate\u2010controlled H2O2generation in defined subcellular compartments of cultured cells. The system exploits the absence of endogenousd\u2010amino acid substrates in mammalian cells ensuring that DAAO\u2010driven H2O2production is activated only upon exogenousd\u2010alanine addition. This approach serves as the conceptual foundation for the HyPer7\u2010DAAO fusion construct and thed\u2010alanine stimulation paradigm described in this article."}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70430","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70430","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70430","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:15:50Z","timestamp":1787739350000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70430"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8]]},"references-count":28,"journal-issue":{"issue":"8","published-print":{"date-parts":[[2026,8]]}},"alternative-id":["10.1002\/cpz1.70430"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70430","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8]]},"article-number":"e70430"}}