TY - JOUR
T1 - Analysis and optimization of conditions for the use of 2′,7′-dichlorofluorescein diacetate in cultured hepatocytes
AU - Reiniers, Megan J.
AU - de Haan, Lianne R.
AU - Reeskamp, Laurens F.
AU - Broekgaarden, Mans
AU - van Golen, Rowan F.
AU - Heger, Michal
N1 - Funding Information: Funding: M.H. was supported by grants from the Dutch Cancer Foundation (KWF project # 10666), a Zhejiang Provincial Foreign Expert Program Grant, Zhejiang Provincial Key Natural Science Foundation of China (#Z20H160031), and a grant for the establishment of the Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics. M.H. is chief formulation officer at Nurish.Me and Camelina Sun and has equity in those companies (whose business activities are unrelated to the present work). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/5/1
Y1 - 2021/5/1
N2 - Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2 ′,7 ′-dichlorodihydrofluorescein-diacetate (DCFH 2-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 2-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 2-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 2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH 2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH 2-DA in cell culture medium relied on medium composition. The use of DCFH 2-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.
AB - Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2 ′,7 ′-dichlorodihydrofluorescein-diacetate (DCFH 2-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 2-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 2-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 2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH 2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH 2-DA in cell culture medium relied on medium composition. The use of DCFH 2-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.
KW - Anoxia and reoxygenation
KW - Cellular uptake and export
KW - Fluorogenic redox probe
KW - Hepatocytes
KW - Intravital fluorescence imaging
KW - Liver diseases
KW - Oxidative and nitrosative stress
UR - http://www.scopus.com/inward/record.url?scp=85104571701&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/antiox10050674
DO - https://doi.org/10.3390/antiox10050674
M3 - Article
C2 - 33925917
SN - 2076-3921
VL - 10
JO - Antioxidants
JF - Antioxidants
IS - 5
M1 - 674
ER -