Mgcl2

Pentraxin 3 binding to TSP1 was performed as previously described (Deban et al, 2010). Briefly 96‐well plates (Nunc Maxisorb immunoplates, Roskilde, Denmark) were coated overnight with TSP1 (39–78 nM) in phosphate buffer (PBS⁺⁺; contains 130 mg/l (1.2 mM) CaCl₂ and 100 mg/l (1.4 mM) MgCl₂; Lonza). After blocking of non‐specific sites with 0.5% dry milk in PBS⁺⁺ (2 h at room temperature), plates are incubated with 100 μl of PTX3 (1.4–220 nM considering a molecular weight of 45 kDa for the PTX3 monomer) in PBS⁺⁺ containing 0.05% Tween 20 (PBST). After washing, plates were first incubated with rabbit anti‐PTX3 polyclonal antibody (1:2,000) and then with anti‐rabbit‐IgG labeled with horseradish peroxidase (HRP: GE Healthcare, Pittsburgh, PA, USA). The chromogen substrate 3′,5,5′‐tetramethylbenzidine (TMB; 1 Step™ ULTRA TMB‐ELISA, Thermo Scientific, Rockford, IL, USA) was added and stopped with 2 N H₂SO₄ before reading absorbance at 450 nm. Binding to immobilized recombinant TSP‐2 and TSP‐4 and to the TSP1 fragments C‐term; N‐term; Ca‐1; P123‐1; E123‐1; and E123CaG‐1 all used at 50 nM, was performed following the same procedure. PTX3 N‐terminal domain (8.8–220 nM) was also tested in the same setting.