Griess reagent

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).

Aortic segments or HUVECs were treated with high glucose (HG; 30 mM) and JAT (1 µM) in a 24-well plate for 48 h, and the supernatants were collected to detect the amount of NO by Griess reagent (Invitrogen, Oregon, USA) according to the manufacturer’s instructions. The SpectraMax M5 microplate reader (Molecular Devices, Silicon Valley, CA, USA) was used to read absorbance at 548 nm.

RAW264.7 cells were seeded into 24-well plates at a density of 2.5×104 cells/well. After nt-p65-TMD or LY294002 treatment in dose-dependent manner for 2 h, the cells were incubated with LPS (0.1 µg/mL) for 20 h at 37 °C in CO₂ incubator. To determine the nitrite release in the culture media, presumed to reflect the NO levels, Griess reaction was used, wherein 100 µL cell culture medium was mixed with 100 µL Griess reagent (Invitrogen) and incubated at room temperature for 30 min. The NO concentration was determined at 540 nm using a spectrophotometer (Bio-Rad Laboratories Inc., Hercules, CA, USA)

The viability and NO production of sister cultures of the cells were determined using the MTT assay (DOJINDO, Tokyo, Japan) and Griess reagent (Invitrogen; Carlsbad, CA, USA), respectively. The generation of NO was determined by measuring the nitrite accumulation in the medium with modified Griess reagent. The culture supernatant and Griess reagent were mixed and incubated for 5 min, and subsequently, the absorption was determined at 540 nm. Sodium nitrite (NaNO₂) was used to generate a standard curve for quantification [28] (link), [29] (link).