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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Implications of dichlorofluorescein photoinstability for detection of UVA-induced oxidative stress in fibroblasts and keratinocyte cells

Full text
Author(s):
Souza, Carla [1] ; Monico, Danielli Azevedo [1] ; Tedesco, Antonio Claudio [1]
Total Authors: 3
Affiliation:
[1] Univ Sao Paulo, Sch Philosophy Sci & Literature Ribeirao Preto, Ctr Nanotechnol & Tissue Engn, Chem Dept, Photobiol & Photomed Res Grp, BR-14040901 Sao Paulo - Brazil
Total Affiliations: 1
Document type: Journal article
Source: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 19, n. 1, p. 40-48, JAN 1 2020.
Web of Science Citations: 0
Abstract

Although the dichlorofluorescein (DCF) assay is widely used to detect the production of UVA-induced ROS, the photostability and phototoxicity of the probe after UVA irradiation remains controversial and the experimental conditions often vary across studies, making it difficult to compare results from different studies. This study aimed to evaluate the suitability of the DCF assay for detection of UVA-induced ROS in human cells after UVA irradiation. Human primary fibroblasts (HPF) and HaCaT cells were loaded with 2 `,7 `-dichlorodihydrofluorescein diacetate (DCFDA) (2, 10, and 50 mu M) for 10 and 30 min, before and after exposure to UVA radiation (5-50 J cm(-2)). Fluorescence was recorded immediately or 30 min after irradiation using three different techniques: microplate reading, flow cytometry, and confocal scanning microscopy. Cell viability was assessed by flow cytometry before and after UVA exposure. A UVA-dose-dependent increase in ROS was observed at 5-50 mu M DCFDA, and the magnitude of the fluorescent signal was affected by RPMI medium, as well as DCFDA loading concentration and incubation period. However, higher concentrations of DCFDA compromised the viability of both HaCaT and HPF cells after UVA irradiation. The most sensitive and reliable combination for the ROS assay was pre-incubation with 10 mu M DCFDA for 30 min in PBS. Reading the fluorescence 30 min after UVA irradiation diminished the emission signal, as did the DCFDA post-incubation. In conclusion, this single-point DCF assay allowed reproducible and sensitive UVA-induced ROS detection in HaCaT and HPF cells without compromising the cell viability or morphology. (AU)

FAPESP's process: 13/50181-1 - Use of drugs containing nanocarriers with photosensitizers and/or other active compounds applied to cell therapy and treatment of central nervous system disorders
Grantee:Antonio Claudio Tedesco
Support Opportunities: Research Projects - Thematic Grants
FAPESP's process: 17/01272-5 - In vitro reconstituted skin models: development, damage assessment and photoprotective efficacy in the UVA and visible spectral ranges
Grantee:Carla Souza
Support Opportunities: Scholarships in Brazil - Post-Doctoral