These values were averaged for each photograph and the relative fluorescence of all photographs was averaged for each replicate. Mean fluorescence for wounded and sham-wounded samples incubated without DCFH-DA were subtracted from the values for samples incubated with DCFH-DA to correct for background fluorescence. When 10% of replicates were re-measured, they were on average

<5% different from the original measured value. Cellular accumulation of strong oxidants after grazing was determined for P. decipiens, the only species of macroalgae AP24534 included in this study that is palatable to the amphipod G. antarctica, by measuring the oxidation of DCFH in vivo. Circular, paired P. decipiens samples (n = 5, 8–10 mm diameter) were excised with a cork borer 24–48 h prior to experimentation and held in flowing seawater until use. Both samples from each individual were placed in a plastic bottle containing five G. antarctica in filtered seawater. Bottles were floated in an aquarium containing flow-through ambient seawater (~1.5°C) for 2 h to allow grazing. Samples were removed from grazers and one sample was incubated

with DCFH-DA, while its paired sample was incubated in the same manner, but without DCFH-DA, as described previously. Samples were viewed microscopically and imaged according to the previous section with the exception that three photographs were taken of each sample, each photograph containing Talazoparib concentration a haphazardly chosen section of grazed

edge with a substantial portion of inner, ungrazed thallus. Since G. antarctica feed on edges (authors’ personal observations), photographs were analyzed for brightness using ImageJ as above, but the five randomly determined sections analyzed for grazed tissue were chosen from directly along the grazed thallus edge and the five randomly determined sections analyzed for control tissue were chosen from directly along the side of the photograph nearest the inner, ungrazed thallus. Strong oxidants in the seawater medium were quantified medchemexpress by measuring the oxidation of DCFH in the presence of esterase and peroxidase using a fluorometer (following Weinberger et al. 1999) before and 1 min after wounding. Paired samples of A. mirabilis, H. grandifolius, T. antarcticus (n = 10), and D. anceps and P. decipiens (n = 9) were placed in 15 mL SFSW under constant rotation on ice. Samples were wounded by multiple punches using a sterile plastic 5 mL pipette tip over the entire thallus surface. Paired, control tissue was treated in the same way as wounded samples with the exception of wounding. To sample, 1,000 μL of the seawater medium was combined with either 500 U catalase (Sigma C9322) in DI or the same volume of DI water.