Mouse cd14

To determine whether Gal-8 binds to LPS, wells of 96-well flat bottom plates (Costar) were coated with rGal-8, (2.5 - 20 μg/mL PBS, 2h, room temp), nonspecific binding sites were blocked with 1% BSA in PBS (1h, room temp.). Biotinylated-LPS (Invitrogen, 50 μl, 1 μg/mL PBS containing 1% BSA) was added to the galectin-coated wells, plates were incubated at room temperature for 1h, and then washed with PBS containing 0.05% Tween 20. Streptavidin-HRP (50 μl, 1:40 dilution, R&D Systems) was added to each well, plates were incubated for 20 min. washed, and air dried. Tetramethylbenzidine (TMB) chromogenic substrate (50 μl/well, R&D) was added to each well for HRP detection, reactions were stopped by 1N H₂SO₄ (50 μl/well), and plates were read in a microplate reader (FilterMax F5 multi-mode, Molecular Devices). Wells coated with mouse CD14 (10 ng/mL, R&D Systems), a well-known LPS-binding protein, served as a positive control. In some experiments, wells were coated with Gal-3 for comparison purposes.