Quantification of MPO-DNA Complexes in Sepsis

An in-house ELISA was used to quantify MPO-DNA complexes in plasma and peritoneal fluid from sham and CLP mice 24 hours after the surgical procedure (24 (link), 27 (link)). Briefly, after overnight coating with anti-MPO capture antibody in calcium carbonate coating buffer (2 µg/ml; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% bovine serum albumin in PBS for 2 hours at room temperature. The plate was subsequently washed, before incubating for 90 minutes at room temperature with 10% peritoneal fluid in blocking buffer. The plate was washed five times, and then incubated for 90 minutes at room temperature with anti-DNA detection antibody (1:20; Cell Death detection ELISA, 11544675001, Sigma). After five washes, the plate was developed with TMB substrate solution (Sigma).