Standard
Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. / Reiniers, Megan J.; de Haan, Lianne R.; Reeskamp, Laurens F. et al.
Methods in Molecular Biology. Vol. 2451 Humana Press Inc., 2022. p. 721-747 (Methods in Molecular Biology; Vol. 2451).
Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review
Harvard
Reiniers, MJ, de Haan, LR, Reeskamp, LF, Broekgaarden, M, Hoekstra, R, van Golen, RF 2022, Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. in Methods in Molecular Biology. vol. 2451, Methods in Molecular Biology, vol. 2451, Humana Press Inc., pp. 721-747. https://doi.org/10.1007/978-1-0716-2099-1_39
APA
Reiniers, M. J., de Haan, L. R., Reeskamp, L. F., Broekgaarden, M., Hoekstra, R., van Golen, R. F. (2022). Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. In Methods in Molecular Biology (Vol. 2451, pp. 721-747). (Methods in Molecular Biology; Vol. 2451). Humana Press Inc.. https://doi.org/10.1007/978-1-0716-2099-1_39
Vancouver
Reiniers MJ, de Haan LR, Reeskamp LF, Broekgaarden M, Hoekstra R, van Golen RF et al. Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. In Methods in Molecular Biology. Vol. 2451. Humana Press Inc. 2022. p. 721-747. (Methods in Molecular Biology). doi: 10.1007/978-1-0716-2099-1_39
Author
Reiniers, Megan J. ; de Haan, Lianne R. ; Reeskamp, Laurens F. et al. / Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes. Methods in Molecular Biology. Vol. 2451 Humana Press Inc., 2022. pp. 721-747 (Methods in Molecular Biology).
BibTeX
@inbook{638e9f4bf90b461f9f6be586828c9a67,
title = "Optimal Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes",
abstract = "Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell{\textquoteright}s capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2′,7′-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2′,7′-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2′,7′-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.",
keywords = "2′,7′-Dichlorodihydrofluorescein-diacetate, 2′,7′-Dichlorofluorescein, Cellular uptake and efflux, Fluorogenic redox probe, Hepatocytes, Oxidative and nitrosative stress",
author = "Reiniers, {Megan J.} and {de Haan}, {Lianne R.} and Reeskamp, {Laurens F.} and Mans Broekgaarden and Ruurdtje Hoekstra and {van Golen}, {Rowan F.} and Michal Heger",
note = "Publisher Copyright: {\textcopyright} 2022, Springer Science+Business Media, LLC, part of Springer Nature.",
year = "2022",
doi = "10.1007/978-1-0716-2099-1_39",
language = "English",
volume = "2451",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "721--747",
booktitle = "Methods in Molecular Biology",
}
RIS
TY - CHAP
T1 - Optimal 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 - Hoekstra, Ruurdtje
AU - van Golen, Rowan F.
AU - Heger, Michal
N1 - Publisher Copyright:© 2022, Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022
Y1 - 2022
N2 - Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell’s capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2′,7′-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2′,7′-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2′,7′-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.
AB - Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell’s capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2′,7′-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2′,7′-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2′,7′-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.
KW - 2′,7′-Dichlorodihydrofluorescein-diacetate
KW - 2′,7′-Dichlorofluorescein
KW - Cellular uptake and efflux
KW - Fluorogenic redox probe
KW - Hepatocytes
KW - Oxidative and nitrosative stress
UR - http://www.scopus.com/inward/record.url?scp=85129572313&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-2099-1_39
DO - 10.1007/978-1-0716-2099-1_39
M3 - Chapter
C2 - 35505044
VL - 2451
T3 - Methods in Molecular Biology
SP - 721
EP - 747
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -