Fluorimetric assay kit

The nitrate/nitrite (NOx) accumulation in the culture medium of HepG2 cells (~2.5 × 10⁵ cells/mL) was measured after OP and NP treatment as previously described. After incubation, the cell supernatants were centrifuged at 4°C, 1000 ×g for 10 min, and the NOx content was measured at the Fluorescence Plate Reader VICTOR Multilabel Counter (Perkin Elmer) using the fluorescent probe DAN (2,3-diaminonaphthalene) (Fluorimetric Assay Kit, Cayman Chemical) [72 (link)].

Cells were incubated with different test reagents for 24 h and then washed with PBS. The supernatant was used for the measurement of nitrite and nitrate with a fluorimetric assay kit (Cat # 780051, Cayman Chemical Company, Ann Arbor, MI, USA) based on the Greiss reaction. The assay is based on the enzymatic conversion of nitrate to nitrite by nitrate reductase followed by the addition of 2,3-diaminonaphthalene, which converts nitrite to a fluorescent compound. Fluorescence intensity measurements of this compound accurately determine the nitrite (NO₂) concentration (excitation max.: 365 nm; emission max.: 450 nm).

Acid sphingomyelinase activity was measured using a commercial fluorimetric assay kit (Cayman). Briefly, aSMase was determined in lung homogenate using an aSMase solution provided by the kit (50 mM sodium acetate, pH = 5; 0.01 g of lung/100 μL aSMase solution). After incubation on ice for 15 min, the homogenate was centrifuged (2780 rpm, 4 °C, 10 min). The supernatant was collected and sonicated (5 s) and aSMase activity was analyzed using the fluorometer Varioskan (Thermo Scientific, excitation and emission wavelengths of 535 and 590 nm, respectively). The activity was determined in 36 μg protein and expressed as nmol/min/mL [11 (link)].