Oxidative stress in L. vannamei hemocytes

Description

Oxidative stress can be used by thehost as a defense mechanism to kill invading pathogens. Conversely, it can use be induced by some pathogens in order to damage the host cell. In either case, determination of oxidative stress in cells is an important host health indicator.

Brief protocol

Detection of ROS in live cells

Image-iT Live Green Reaction Oxygen Species Detection Kit (invitrogen) is used to estimate the generation of reactive oxygen species in shrimp hemocyte. Expose the seeded shrimp hemocyte monolayers to carboxy-H2DCFDA (25µM in 2x L-15 medium) for 30 min. After counterstaining the nucleus with DAPI (4'-6-Diamidino-2-phenylindole) for 5 min, the cells were washed twice again and fluorescent signals were viewed with a microscope. Data were subjected to unpaired Student’s t-tests. P values of less than 0.05 were considered to be a statistically significant difference. tert-butyl hydroperoxide (TBHP) (100µM in 2x L-15 medium) expose to the cell for 30 min before carboxy-H2DCFDA staining, which serve as the ROS induction control in this experiment.

Hydrogen peroxide assay

Amplex® Red Hydrogen Peroxide⁄Peroxidase Assay Kit (invitrogen) is used to detect the amount of the H2O2 generated from live cells. Shrimp hemolymphs are collect and centrifuge at 10,000rpm, 4℃ for 3 minutes and then discarded the suspension, the pellets are rinsed one time with cold 1x PBS, carefully suspend with 200 μl 0.33 x PBS and placed on ice for 10 minutes. The cell lysates were centrifuged at 13,000 rpm for 5 minutes again and collected the suspensions to new tubes. The hemocyte are serially diluted with 0.33 x PBS to determine the optimal concentration for the assay. H2O2 standard curve was generated with 0, 0.625, 1.25, 2.5, 5, 10 and 20 μM and carried out the same subsequent steps with shrimp samples. 50 μl hemocyte lysates or standard samples were mixed with 50 μl Amplex Red reagent/HRP working solution, and then placed into a 96 well-plate. Incubate the sample at room temperature for 30 mins, protected form light. The absorbance of the sample is measured at 560 nm with an ELISA reader.