Open Access
Antioxidants, volume 10, issue 5, pages 674
Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
Reiniers Megan J.
1, 2
,
de Haan Lianne R.
1, 3
,
Reeskamp Laurens F.
4
,
Broekgaarden Mans
5, 6
,
van Golen Rowan F.
7
,
Heger M.
1, 3, 8
2
Department of Surgery, Haaglanden Medisch Centrum, 2512 VA The Hague, The Netherlands
|
3
Laboratory of Experimental Oncology, Department of Pathology, Erasmus MC, 3015 GD Rotterdam, The Netherlands
|
4
Department of Vascular Medicine, Amsterdam UMC, Location AMC, 1105 AZ Amsterdam, The Netherlands
|
6
INSERM U 1209, Allée des Alpes, 38700 La Tronche, France
|
7
Department of Gastroenterology and Hepatology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
|
8
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands
|
Publication type: Journal Article
Publication date: 2021-04-26
PubMed ID:
33925917
Biochemistry
Molecular Biology
Cell Biology
Clinical Biochemistry
Physiology
Abstract
Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-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 DCFH2-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. DCFH2-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 DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-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.
Citations by journals
|
1
2
|
|
|
Antioxidants
|
Antioxidants
2 publications, 16.67%
|
|
Molecules
|
Molecules
1 publication, 8.33%
|
|
Frontiers in Endocrinology
|
Frontiers in Endocrinology
1 publication, 8.33%
|
|
Cells
|
Cells
1 publication, 8.33%
|
|
International Journal of Molecular Sciences
|
International Journal of Molecular Sciences
1 publication, 8.33%
|
|
Dalton Transactions
|
Dalton Transactions
1 publication, 8.33%
|
|
Radiation and Environmental Biophysics
|
Radiation and Environmental Biophysics
1 publication, 8.33%
|
|
Microbial Pathogenesis
|
Microbial Pathogenesis
1 publication, 8.33%
|
|
RSC Advances
|
RSC Advances
1 publication, 8.33%
|
|
Journal of Medicinal Chemistry
|
Journal of Medicinal Chemistry
1 publication, 8.33%
|
|
Food Control
|
Food Control
1 publication, 8.33%
|
|
1
2
|
Citations by publishers
|
1
2
3
4
5
|
|
|
Multidisciplinary Digital Publishing Institute (MDPI)
|
Multidisciplinary Digital Publishing Institute (MDPI)
5 publications, 41.67%
|
|
Royal Society of Chemistry (RSC)
|
Royal Society of Chemistry (RSC)
2 publications, 16.67%
|
|
Elsevier
|
Elsevier
2 publications, 16.67%
|
|
Frontiers Media S.A.
|
Frontiers Media S.A.
1 publication, 8.33%
|
|
Springer Nature
|
Springer Nature
1 publication, 8.33%
|
|
American Chemical Society (ACS)
|
American Chemical Society (ACS)
1 publication, 8.33%
|
|
1
2
3
4
5
|
- We do not take into account publications that without a DOI.
- Statistics recalculated only for publications connected to researchers, organizations and labs registered on the platform.
- Statistics recalculated weekly.
{"yearsCitations":{"type":"bar","data":{"show":true,"labels":[2021,2022,2023,2024],"ids":[0,0,0,0],"codes":[0,0,0,0],"imageUrls":["","","",""],"datasets":[{"label":"Citations number","data":[1,4,5,2],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":["8.33","33.33","41.67","16.67"],"barThickness":null}]},"options":{"indexAxis":"x","maintainAspectRatio":true,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":1,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Citations per year","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}},"journals":{"type":"bar","data":{"show":true,"labels":["Antioxidants","Molecules","Frontiers in Endocrinology","Cells","International Journal of Molecular Sciences","Dalton Transactions","Radiation and Environmental Biophysics","Microbial Pathogenesis","RSC Advances","Journal of Medicinal Chemistry","Food Control"],"ids":[16381,1770,19896,25293,14627,8473,1136,8685,3100,3673,20338],"codes":[0,0,0,0,0,0,0,0,0,0,0],"imageUrls":["\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/4QWA67eqfcfyOiA8Wk7YnqroHFqQbTsmDJUYTCTg_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/leiAYcRDGTSl5B1eCnwpSGqmDEUEfDPPoYisFGhT_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/leiAYcRDGTSl5B1eCnwpSGqmDEUEfDPPoYisFGhT_medium.webp","\/storage\/images\/resized\/iLiQsFqFaSEx6chlGQ5fbAwF6VYU3WWa08hkss0g_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp"],"datasets":[{"label":"","data":[2,1,1,1,1,1,1,1,1,1,1],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":[16.67,8.33,8.33,8.33,8.33,8.33,8.33,8.33,8.33,8.33,8.33],"barThickness":13}]},"options":{"indexAxis":"y","maintainAspectRatio":false,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":null,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Journals","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}},"publishers":{"type":"bar","data":{"show":true,"labels":["Multidisciplinary Digital Publishing Institute (MDPI)","Royal Society of Chemistry (RSC)","Elsevier","Frontiers Media S.A.","Springer Nature","American Chemical Society (ACS)"],"ids":[202,123,17,208,8,40],"codes":[0,0,0,0,0,0],"imageUrls":["\/storage\/images\/resized\/MjH1ITP7lMYGxeqUZfkt2BnVLgjkk413jwBV97XX_medium.webp","\/storage\/images\/resized\/leiAYcRDGTSl5B1eCnwpSGqmDEUEfDPPoYisFGhT_medium.webp","\/storage\/images\/resized\/GDnYOu1UpMMfMMRV6Aqle4H0YLLsraeD9IP9qScG_medium.webp","\/storage\/images\/resized\/4QWA67eqfcfyOiA8Wk7YnqroHFqQbTsmDJUYTCTg_medium.webp","\/storage\/images\/resized\/voXLqlsvTwv5p3iMQ8Dhs95nqB4AXOG7Taj7G4ra_medium.webp","\/storage\/images\/resized\/iLiQsFqFaSEx6chlGQ5fbAwF6VYU3WWa08hkss0g_medium.webp"],"datasets":[{"label":"","data":[5,2,2,1,1,1],"backgroundColor":["#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6","#3B82F6"],"percentage":[41.67,16.67,16.67,8.33,8.33,8.33],"barThickness":13}]},"options":{"indexAxis":"y","maintainAspectRatio":false,"scales":{"y":{"ticks":{"precision":0,"autoSkip":false,"font":{"family":"Montserrat"},"color":"#000000"}},"x":{"ticks":{"stepSize":null,"precision":0,"font":{"family":"Montserrat"},"color":"#000000"}}},"plugins":{"legend":{"position":"top","labels":{"font":{"family":"Montserrat"},"color":"#000000"}},"title":{"display":true,"text":"Publishers","font":{"size":24,"family":"Montserrat","weight":600},"color":"#000000"}}}}}
Metrics
Cite this
GOST |
RIS |
BibTex |
MLA
Cite this
GOST
Copy
Reiniers M. J. et al. Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes // Antioxidants. 2021. Vol. 10. No. 5. p. 674.
GOST all authors (up to 50)
Copy
Reiniers M. J., de Haan L. R., Reeskamp L. F., Broekgaarden M., van Golen R. F., Heger M. Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes // Antioxidants. 2021. Vol. 10. No. 5. p. 674.
Cite this
RIS
Copy
TY - JOUR
DO - 10.3390/antiox10050674
UR - https://doi.org/10.3390%2Fantiox10050674
TI - Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes
T2 - Antioxidants
AU - de Haan, Lianne R.
AU - Reeskamp, Laurens F.
AU - van Golen, Rowan F.
AU - Reiniers, Megan J.
AU - Broekgaarden, Mans
AU - Heger, M.
PY - 2021
DA - 2021/04/26 00:00:00
PB - Multidisciplinary Digital Publishing Institute (MDPI)
SP - 674
IS - 5
VL - 10
PMID - 33925917
SN - 2076-3921
ER -
Cite this
BibTex
Copy
@article{2021_Reiniers,
author = {Lianne R. de Haan and Laurens F. Reeskamp and Rowan F. van Golen and Megan J. Reiniers and Mans Broekgaarden and M. Heger},
title = {Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes},
journal = {Antioxidants},
year = {2021},
volume = {10},
publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},
month = {apr},
url = {https://doi.org/10.3390%2Fantiox10050674},
number = {5},
pages = {674},
doi = {10.3390/antiox10050674}
}
Cite this
MLA
Copy
Reiniers, Megan J., et al. “Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes.” Antioxidants, vol. 10, no. 5, Apr. 2021, p. 674. https://doi.org/10.3390%2Fantiox10050674.