Plasma samples were analyzed using commercial ELISA kits for mouse C3b (HK216, Hycult Biotech, Uden, The Netherlands), C5a (DY2150; R&D Systems, Minneapolis, MN), hyaluronan (DY3614; R&D Systems), and syndecan-1 (75-138MS-S10; Alpco, Salem, NH), as per the manufacturer's instructions.
Plasma HMGB1 was measured using an in-house ELISA. In brief, a capture antibody, rabbit anti-HMGB1 (H9539; Sigma-Aldrich, Castle Hill, Australia) and detection antibody, HRP-conjugated rabbit anti-HMGB1 (ab128129; Abcam, Melbourne, Australia) were used, followed by SureBlue TMB Microwell Peroxidase Substrate (KPL 52-00-01, SeraCare, Milford, MA). Human HMGB1 full length protein, which shares 99% sequence homology with mouse HMGB1, was used as standard. Optical density was measured at 450/540 nm using a FLUOstar Omega microplate reader (BMG Labtech GmbH, Offenburg, Germany).
Plasma HMGB1 was measured using an in-house ELISA. In brief, a capture antibody, rabbit anti-HMGB1 (H9539; Sigma-Aldrich, Castle Hill, Australia) and detection antibody, HRP-conjugated rabbit anti-HMGB1 (ab128129; Abcam, Melbourne, Australia) were used, followed by SureBlue TMB Microwell Peroxidase Substrate (KPL 52-00-01, SeraCare, Milford, MA). Human HMGB1 full length protein, which shares 99% sequence homology with mouse HMGB1, was used as standard. Optical density was measured at 450/540 nm using a FLUOstar Omega microplate reader (BMG Labtech GmbH, Offenburg, Germany).