2 thiobarbituric acid

The uninfected and vvIBDV-infected spleens and bursae were homogenised, filtered through a 70 μm mesh, and pelleted at 500 xg for 10 min. Supernatants from both the spleen and bursae were subjected to quantification of nitric oxide and malondialdehyde levels. NO was quantified using the Griess assay, where 150 μl of the supernatant was added to 20 μl of Griess reagent (Invitrogen, USA) and 130 μl of deionised water, followed by 30 min incubation at room temperature. The sample’s absorbance was read at 548 nm using a μQuant ELISA Reader (BioTek Instruments, USA). For the malondialdehyde (MDA) determination, 200 μl of supernatant were added to 800 μl of PBS, 25 μl of butylated hydroxytoluene (Sigma, USA), and 500 μl of trichloroacetic acid and 2-thiobarbituric acid (Sigma, USA), followed by 2 h incubation on ice. The mixture was then pelleted at 500 xg for 15 min and 1 ml of the supernatant was collected and added to 75 μl of 0.1 M ethylene diamine tetraacetic acid and 250 μl of 0.05 M 2-thiobarbituric acid (Sigma, USA). Finally, the sample was boiled for 15 min, cooled to room temperature, and the absorbance recorded at 548 nm by the μQuant ELISA Reader. A standard curve for MDA was prepared concurrently using thiobarbituric reactive substances (Sigma, USA).

Copper sulfate, copper bisglycinate, 2-Thiobarbituric acid (TBA), 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB), sulfosalicylic acid, and propionaldehyde diethyl acetal were purchased from Sigma Company (St. Louis, MO, USA). Copper proteinate was obtained from a commercial premix company (Bioplex^®, Alltech Inc., Nicholasville, KY, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) was obtained from Hyclone (Logan, UT, USA). A BCA TM Protein Assay Kit was obtained from Pierce (Rockford, IL, USA). A Protein G agarose column was obtained from Upstate Biotechnology (Lake Placid, NY, USA). ELISA plates (96 wells) and other cell culture plastic wares were obtained from Costar (Cambridge, MA, USA). HPLC grade methanol and acetonitrile were obtained from Fisher Scientific International (Hampton, NH, USA). HPLC grade formic acid and ammonium acetate were purchased from Dima Technology (Richmond Hill, Canada). Water used was ultrapure water (Milli-Q, Millipore Corporation, Billerica, MA, USA).