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In the literature, establishing such models vary widely with regards to cell lines, iron source, iron treatment conditions and duration. Due to this diversity, researchers reported significant differences in the measured outcomes, either in cellular function or response to a stimulus. Herein, we report the process required to establish an iron overload HepG2 cell model to achieve a consistent and reproducible results such that the literature can strive towards a consensus. Iron loading in cells was achieved with 50\u00a0\u03bcM of iron every 24\u00a0h for 2\u00a0days, followed by an additional 24\u00a0h of maintenance in fresh media. We demonstrated that iron overloaded cells had significantly increased ROS generation, labile and total iron whilst having various cellular functions resemble cells without iron overload. The present report addresses key pitfalls with regards to the lack of consensus currently present in the literature.<\/jats:p>","DOI":"10.1038\/s41598-023-49072-7","type":"journal-article","created":{"date-parts":[[2023,12,11]],"date-time":"2023-12-11T10:02:44Z","timestamp":1702288964000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Development of an iron overload HepG2 cell model using ferrous ammonium citrate"],"prefix":"10.1038","volume":"13","author":[{"given":"Usama","family":"Abbasi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Srinivas","family":"Abbina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arshdeep","family":"Gill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jayachandran N.","family":"Kizhakkedathu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2023,12,8]]},"reference":[{"key":"49072_CR1","doi-asserted-by":"publisher","first-page":"750","DOI":"10.1007\/s13238-014-0083-7","volume":"5","author":"C Zhang","year":"2014","unstructured":"Zhang, C. 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