Scheme 2.

NO synthesis was measured in ECV-304 cells cultured with plasma from healthy controls and patients with RA. (a) ECV-304 cells were cultured in 96 well plates before plasma addition. Then, plasma from healthy controls and patients with RA was incubated for 12 h at 37 °C 5% CO₂. Each plate contained a negative control (No DAF-2DA). After 12 h, ECV-304 cells were washed with PBS and cultured with 5 μM DAF-2DA for 30 min at 37 °C 5% CO₂. Fluorescence intensity was then measured using a microplate reader at emission 540 nm (excitation 488 nm). The basal fluorescence intensity was measured before Histamine [10 mM] addition. Then, the plate was activated with Histamine [10 mM] and the fluorescence signal of NO was measured at 60, 180, 300 and 600 s. (b) Fluorescence intensity per sample was normalized by total protein concentration on each well. (c) Fluorescence intensity at 0 s (basal level) was subtracted from fluorescence intensity at 0, 60, 180, 300 and 600 s post-activation with Histamine [10 uM] and tabulated to calculate the kinetics parameters of NO synthesis. Maximum fluorescence intensity (Fmax) and Km for each sample was obtained according to Michaelis-Menten equation. The slope of NO production per sample was obtained by dividing the maximum fluorescence intensity (Fmax) by Km (t_1/2). (d) Fluorescence intensity from Histamine-stimulated ECV-304 cells in the presence of the nitric oxide synthase inhibitor N-nitro l-arginine methyl ester (L-NAME) was used to confirm NO synthesis.