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Implications of using the fluorescent probes, dihydrorhodamine 123 and 2 ',7 '-dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species

Lookup NU author(s): Dr SarahJayne Boulton, Alasdair Anderson, Dr Helen Swalwell, Dr James Henderson, Dr Philip Manning, Professor Mark Birch-Machin

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Abstract

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.


Publication metadata

Author(s): Boulton S, Anderson A, Swalwell H, Henderson JR, Manning P, Birch-Machin MA

Publication type: Article

Publication status: Published

Journal: Free Radical Research

Year: 2011

Volume: 45

Issue: 2

Pages: 115-122

Print publication date: 01/02/2011

ISSN (print): 1071-5762

ISSN (electronic): 1029-2470

Publisher: Informa Healthcare

URL: http://dx.doi.org/10.3109/10715762.2010.517751

DOI: 10.3109/10715762.2010.517751


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