Autor(es)

Souza, Carla ^[1] ; Monico, Danielli Azevedo ^[1] ; Tedesco, Antonio Claudio ^[1]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 19, n. 1, p. 40-48, JAN 1 2020.
• Citações Web of Science: 0

Resumo

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)

• Processo FAPESP: 13/50181-1 - Utilização de nanocarreadores contendo fármacos fotossensibilizantes e outros ativos aplicados à terapia celular e tratamento de patologias do sistema nervoso central
• Beneficiário: Antonio Claudio Tedesco
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 17/01272-5 - Modelos in vitro de pele reconstituída: desenvolvimento, avaliação dos danos e da eficácia fotoprotetora nas regiões UVA e visível do espectro solar
• Beneficiário: Carla Souza
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado