Ex Vivo Complement Activation Post-TBI

The LP activity was measured in mouse plasma samples obtained at 4 days after TBI or sham injury. For ex vivo LP assessment, EDTA-plasma samples (2.5% final plasma concentration) were incubated on mannan-coated ELISA plates for 15 min to initiate activation followed by detection of C3c deposition as described elsewhere [22 (link)]. Briefly, EDTA-plasma samples were thawed on ice and suspended in barbital buffered saline (BBS; 4 mM barbital, 145 mMNaCl, 2 mM CaCl₂, 1mM MgCl₂, pH 7.4), to a final plasma concentration of 2.5%. Plasma solutions were incubated on the coated plate at 37 °C for 15 min. The plate was washed and incubated for 1 h 30 min at RT with a polyclonal anti-C3c antibody (Dako, A0062) diluted 1:5000 in washing buffer. After washing, the plate was incubated with an alkaline-phosphatase labelled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:5000 in washing buffer for 1 h 30 min at RT. Following washing, the assay was developed by adding 100 µL substrate solution (Sigma Fast p-Nitrophenyl Phosphate tablets, Sigma) and the absorption at 405 nm measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).