Limulus amebocyte lysate assay kit

The limulus amebocyte lysate assay kit (Cambrex) was used to measure plasma LPS levels, and commercially available ELISA kits were used to assay plasma levels of sCD14 (R&D Systems), C-reactive protein (CRP; R&D Systems) and D-dimer (Imuclone). Custom designed multiplex chemiluminescent ELISA plates (Meso Scale Discovery) were used to assay cytokine and chemokine levels.

Oenothein B was isolated and identified as described previously [4 (link)]. Briefly, fully blossomed E. angustifolium was collected and the dried plant material (400 g) was extracted with 80% methanol at room temperature for 3 days. The combined extracts were concentrated, and any precipitates were removed by filtration through a 0.22-μm filter. The filtrate was lyophilized to obtain the crude extract. The crude extract was re-dissolved and fractionated on a Sephadex LH-20 column (2.8 × 33 cm) using 80% methanol as an eluent. Fractions were collected, pooled based on elution profile (absorbance at 270 nm), evaporated to dryness, and re-chromatographed twice. The appropriate fractions for collection and pooling were identified as previously described [4 (link)]. The identity of the purified compound was confirmed by NMR and mass spectrometry, as described [4 (link)]. Purity was determined to be >95% by HPLC and mass spectrometry. A Limulus amebocyte lysate assay kit (Cambrex, East Rutherford, NJ, USA) was used to evaluate possible endotoxin contamination in purified oenothein B. Purified, endotoxin-free oenothein B was resuspended in Dulbecco’s PBS and stored at −80° C until use in the functional assays described below.