Analysis and Optimization of Conditions for the Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Overview

Journal Antioxidants (Basel)

Date 2021 Apr 30

PMID 33925917

Citations 8

Authors

Megan J Reiniers

Lianne R de Haan

Laurens F Reeskamp

Mans Broekgaarden

Rowan F van Golen

Michal Heger

Affiliations

Soon will be listed here.

Abstract

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH-DA in cell culture medium relied on medium composition. The use of DCFH-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

Citing Articles

Enhancement of 5-Aminolevulinic Acid-Mediated Photodynamic Inactivation of Using Phosphoric and Bisaminophosphinic Acids as Permeabilizing Agents.

Zdubek A, Maliszewska I, Grabowiecka A, Kowalczyk R, Turek B ACS Omega. 2024; 9(49):48629-48641.

PMID: 39676993 PMC: 11635689. DOI: 10.1021/acsomega.4c07696.

Increased radical scavenging activity of thymoquinone and l-ascorbic acid dual encapsulated in palmitoyl-chitosan nanoparticles in a human normal lung fibroblast, MRC-5 due to synergistic antioxidative effects.

Othman N, Md Jamil S, Masarudin M, Jusoh R, Alamassi M RSC Adv. 2023; 13(40):27965-27983.

PMID: 37736560 PMC: 10510457. DOI: 10.1039/d3ra04326f.

On the Photo-Eradication of Methicillin-Resistant Biofilm Using Methylene Blue.

Maliszewska I, Zdubek A Int J Mol Sci. 2023; 24(1).

PMID: 36614237 PMC: 9821080. DOI: 10.3390/ijms24010791.

Anti-hyperglycemic contours of Madhugrit are robustly translated in the model of lipid accumulation by regulating oxidative stress and inflammatory response.

Balkrishna A, Gohel V, Pathak N, Tomer M, Rawat M, Dev R Front Endocrinol (Lausanne). 2022; 13:1064532.

PMID: 36545334 PMC: 9762483. DOI: 10.3389/fendo.2022.1064532.

An Evaluation of the Novel Biological Properties of Diterpenes Isolated from Codd. In Vitro and In Silico.

Sitarek P, Kowalczyk T, Synowiec E, Merecz-Sadowska A, Bangay G, Princiotto S Cells. 2022; 11(20).

PMID: 36291112 PMC: 9600095. DOI: 10.3390/cells11203243.

References

Gomes A, Fernandes E, Lima J . Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods. 2005; 65(2-3):45-80. DOI: 10.1016/j.jbbm.2005.10.003. View

Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I . Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A. 2002; 99(24):15655-60. PMC: 137772. DOI: 10.1073/pnas.232137699. View

Myhre O, Andersen J, Aarnes H, Fonnum F . Evaluation of the probes 2',7'-dichlorofluorescin diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol. 2003; 65(10):1575-82. DOI: 10.1016/s0006-2952(03)00083-2. View

van Raath M, Weijer R, Nguyen G, Choi B, de Kroon A, Heger M . Tranexamic Acid-Encapsulating Thermosensitive Liposomes for Site-Specific Pharmaco-Laser Therapy of Port Wine Stains. J Biomed Nanotechnol. 2018; 12(8):1617-40. PMC: 5457158. DOI: 10.1166/jbn.2016.2277. View

Wrona M, Wardman P . Properties of the radical intermediate obtained on oxidation of 2',7'-dichlorodihydrofluorescein, a probe for oxidative stress. Free Radic Biol Med. 2006; 41(4):657-67. DOI: 10.1016/j.freeradbiomed.2006.05.006. View

Rota C, Chignell C, Mason R . Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic Biol Med. 1999; 27(7-8):873-81. DOI: 10.1016/s0891-5849(99)00137-9. View

Chen X, Zhong Z, Xu Z, Chen L, Wang Y . 2',7'-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radic Res. 2010; 44(6):587-604. DOI: 10.3109/10715761003709802. View

Karlsson M, Kurz T, Brunk U, Nilsson S, Frennesson C . What does the commonly used DCF test for oxidative stress really show?. Biochem J. 2010; 428(2):183-90. DOI: 10.1042/BJ20100208. View

Grzelak A, Rychlik B, Bartosz G . Light-dependent generation of reactive oxygen species in cell culture media. Free Radic Biol Med. 2001; 30(12):1418-25. DOI: 10.1016/s0891-5849(01)00545-7. View

10.

Keynes R, Griffiths C, Garthwaite J . Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments. Biochem J. 2002; 369(Pt 2):399-406. PMC: 1223083. DOI: 10.1042/BJ20020933. View

11.

Wardman P, Candeias L . Fenton chemistry: an introduction. Radiat Res. 1996; 145(5):523-31. View

12.

Du Y, Han R, Ng S, Ni J, Sun W, Wohland T . Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum. Tissue Eng. 2007; 13(7):1455-68. DOI: 10.1089/ten.2006.0381. View

13.

Chouchani E, Pell V, Gaude E, Aksentijevic D, Sundier S, Robb E . Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature. 2014; 515(7527):431-435. PMC: 4255242. DOI: 10.1038/nature13909. View

14.

Hua Long L, Halliwell B . Oxidation and generation of hydrogen peroxide by thiol compounds in commonly used cell culture media. Biochem Biophys Res Commun. 2001; 286(5):991-4. DOI: 10.1006/bbrc.2001.5514. View

15.

Kirsch M, Lomonosova E, Korth H, Sustmann R, De Groot H . Hydrogen peroxide formation by reaction of peroxynitrite with HEPES and related tertiary amines. Implications for a general mechanism. J Biol Chem. 1998; 273(21):12716-24. DOI: 10.1074/jbc.273.21.12716. View

16.

Hart S, Li Y, Nakamoto K, Subileau E, Steen D, Zhong X . A comparison of whole genome gene expression profiles of HepaRG cells and HepG2 cells to primary human hepatocytes and human liver tissues. Drug Metab Dispos. 2010; 38(6):988-94. PMC: 2879958. DOI: 10.1124/dmd.109.031831. View

17.

Baker C, Mock N, Roberts D, Deahl K, Hapeman C, Schmidt W . Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase. Free Radic Biol Med. 2007; 43(9):1322-7. DOI: 10.1016/j.freeradbiomed.2007.07.020. View

18.

Zamek-Gliszczynski M, Xiong H, Patel N, Turncliff R, Pollack G, Brouwer K . Pharmacokinetics of 5 (and 6)-carboxy-2',7'-dichlorofluorescein and its diacetate promoiety in the liver. J Pharmacol Exp Ther. 2003; 304(2):801-9. DOI: 10.1124/jpet.102.044107. View

19.

Zhu H, Bannenberg G, Moldeus P, Shertzer H . Oxidation pathways for the intracellular probe 2',7'-dichlorofluorescein. Arch Toxicol. 1994; 68(9):582-7. DOI: 10.1007/s002040050118. View

20.

Bedard K, Krause K . The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007; 87(1):245-313. DOI: 10.1152/physrev.00044.2005. View