UMB 3: Workshop on The Use of Fluorescent Probes for The Detection of Reactive Oxygen Species (ROS)
T. Gomes (NIVA) and K.E. Tollefsen (NIVA/NMBU-IMV)
Objective: A three-day workshop was held at NIVA on the use of fluorescent probes for the detection of ROS, which was opened to all CERAD partners. The aim of the workshop was to disseminate the knowledge on the methods available at NIVA to detect ROS and form a discussion group for future debate on the applicability of the methods in species of interest.
Description: A total of 18 participants from different institutions attended the workshop. A general introduction on the chemistry of different ROS formation and their detection was given in the first day, followed by a general discussion on how to incorporate and expand the knowledge platform on ROS and other oxidative stress related endpoints within CERAD groups. Afterwards, the protocols already established at NIVA were fully explained and demonstrated in two species (crustacean Daphnia magna and algae Chlamydomonas reinhardtii), focusing on the several steps necessary to develop and adapt a ROS formation protocol to a whole organism vs single cells. After a discussion about the key points to set up an experimental design to develop a ROS bioassay, participants had the chance to develop and test the protocols hands-on in the NIVA laboratory using their species of interest. Several know ROS inducers were used to check the applicability of two probes (H2DCFDA and DHR 123) in the detection of ROS formation in zebrafish Danio rerio embryos and ZF-L cell line (liver cell model), nematodes Caenorhabditis elegans, Atlantic salmon Salmo salar eggs and copepod Acartia tonsa. The results obtained for all species were promising, showing the applicability of using this type of bioassay in different organisms. Nevertheless, methods still need to be further developed and optimized to better reflect ROS formation based on the type of exposure conditions, most sensitive probe and species considered.
Project Manager: Tânia Gomes
Detection of ROS in zebrafish larva exposed to UV, exhibiting increased fluorescence from lowest to highest dose (A-C), while controls showed minimal fluorescence (D-F). (Figure: T. Gomes, NIVA).