Pe conjugated rat anti mouse siglec f

Eosinophils were cultured from BM of naïve mice (on soy-free diet) as described previously [41 (link), 42 (link)]. Differentiated cells were evaluated for expression of MBP by confocal microscopy using rat mAb against murine MBP (2.5 μg/ml) followed by FITC-conjugated goat anti-rat IgG and of Siglec-F by flow cytometry (FACScan and CellQuest Pro^™ Software, BD Biosciences, San Diego, CA) with PE-conjugated rat anti-mouse Siglec-F (5 μg/ml, BD Biosciences). Cells between days 12–14 of culture that were 99% Hema 3-positive and expressed both MBP and Siglec-F were used in studies.
To examine the effect of t-TUCB on signaling events, eosinophils on day 12 of culture were harvested and treated with t-TUCB (5 μM) or DMSO (vehicle control) in PBS for 1, 10 or 30 min at 37°C and processed for Western blot analysis (described below). The effect of inflammatory mediators on expression of sEH was examined by culturing cells (~5 × 10⁶/well) in medium containing 10% FBS and IL-4, TNFα (at 100 ng/ml) or eotaxin-1 (at 100 nM, all from PeproTech, Rocky Hill, NJ) for 24 h at 37°C and then evaluating sEH by Western blot analysis.