Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro.
HepG2
In vitro cytotoxicity
Neutral red uptake
Viability assay
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
medline:
8
5
2023
pubmed:
5
5
2023
entrez:
4
5
2023
Statut:
ppublish
Résumé
The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.
Identifiants
pubmed: 37142926
doi: 10.1007/978-1-0716-3052-5_15
doi:
Substances chimiques
Neutral Red
261QK3SSBH
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
237-245Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Références
Borenfreund E, Puerner J (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR90). J Tissue Cult Methods 9(1):7–9
doi: 10.1007/BF01666038
Repetto G, del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3(7):1125–1131
doi: 10.1038/nprot.2008.75
pubmed: 18600217
Aslantürk ÖS (2018) In vitro cytotoxicity and cell viability assays: principles, advantages, and disadvantages. In Genotoxicity - a predictable risk to our actual world
Zuang V (2001) The neutral red release assay: a review. Altern Lab Anim 29(5):575–599
doi: 10.1177/026119290102900513
pubmed: 11604100
Rodrigues RM, Bouhifd M, Bories G, Sacco M, Gribaldo L, Fabbri M, Coecke S, Whelan MP (2013) Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells. Toxicol In Vitro 27(2):760–767
doi: 10.1016/j.tiv.2012.12.004
pubmed: 23261643
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (2003) Test method protocol for the BALB/c 3T3 neutral red uptake cytotoxicity test. A test for basal cytotoxicity for an in vitro validation study phase III. Available through: https://ntp.niehs.nih.gov/iccvam/methods/acutetox/invidocs/phiiiprot/3t3phiii.pdf . Consulted May 2022
Organisation for Economic Co-operation and Development (2004) Guideline 432: in vitro 3T3 NRU phototoxicity test. OECD guidelines for the testing of chemicals Available through: http://wwwoecd-ilibraryorg/environment/test-no-432-in-vitro-3t3-nru-phototoxicity-test_9789264071162-en . Consulted May 2022
European Commission Joint Research Center (2013) EURL ECVAM recommendation on the 3T3 neutral red uptake (3T3 NRU) cytotoxicity assay for the identification of substances not requiring classification for acute oral toxicity. Available through: https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations/3t3-nru-recommendation . Consulted May 2022
Bouhifd M, Bories G, Casado J, Coecke S, Norlén H, Parissis N, Rodrigues RM, Whelan MP (2012) Automation of an in vitro cytotoxicity assay used to estimate starting doses in acute oral systemic toxicity tests. Food Chem Toxicol 50(6):2084–2096
doi: 10.1016/j.fct.2012.03.046
pubmed: 22465836
Knowles BB, Aden DP (1983) Human hepatoma derived cell line, process for preparation thereof, and uses therefor. US Patent 4,393,133
Schoonen WG, Westerink WM, de Roos JA, Débiton E (2005) Cytotoxic effects of 100 reference compounds on HepG2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake. Toxicol In Vitro 19(4):505–516
doi: 10.1016/j.tiv.2005.01.003
pubmed: 15826808
Chiu JH, Hu CP, Lui WY, Lo SJ, Chang CM (1990) The formation of bile canaliculi in human hepatoma-cell lines. Hepatology 11:834–842
doi: 10.1002/hep.1840110519
pubmed: 2161394
Hewitt NJ, Hewitt P (2004) Phase I and II enzyme characterization of two sources of HepG2 cell lines. Xenobiotica 34(3):243–256
doi: 10.1080/00498250310001657568
pubmed: 15204697