Vacutainer ssttm tubes

Cell culture supernatants were purified by centrifugation for 20 min at 3000 r.p.m and stored at −80 °C. Serum was separated from the venous blood using the BD Vacutainer SST^TM tubes (BD Biosciences, San Jose, CA, USA) after centrifugation for 20 min at 3000 r.p.m and stored at −80 °C. CCL5 and CXCL12 levels were detected in duplicate assays using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols.

CHI-induced neurological impairment was assessed using the NSS. Detailed methods were provided in Supplementary Table S2. In addition, animals were sacrificed after anaesthesia and their fresh or perfused-fixed brain tissues were collected. Cerebral edema and BBB permeability were measured by two blinded, trained investigators as previously described23 (link). For analysis of serum complement content, mouse blood harvested via cardiac puncture immediately prior to perfusion was transferred to sterile BD Vacutainer SST^TM tubes (BD Biosciences, San Jose, CA, USA) and centrifuged at 3,000 rpm at 4 °C (20 min). The supernatants were collected and stored at −80 °C.

CHI mouse serum was collected at 12 h post-trauma as previously described^{22 (link)}. Briefly, blood was harvested via cardiac puncture. Blood was transferred to sterile BD Vacutainer SST^TM tubes (BD Biosciences, San Jose, CA, USA) and subsequently centrifuged at 3000 rpm at 4 °C (20 min). The supernatants were collected and stored at –80 °C. HI-CHI mouse serum was processed by heating to 56 °C for 45 min^{44 (link)}.