Mice between 5–7 wk were used for in vivo experiments comparing BALBc mice (000651; The Jackson Laboratory) and C3ar1tm1Cge knock-out mice (C.129S4-C3ar1tm1cge/J; The Jackson Laboratory). All mouse studies were approved by the institutional animal care and use committees of Stanford University.
In mice tracked for temperature and weight, 1 wk before sepsis, mice were anesthetized locally with 1% lidocaine (4 mg/kg) on the right side hind-back using a 0.5 ml syringe. After 15 min, electronic temperature and ID transponders were implanted subcutaneously in the same right side hind-back region (IPTT-300 transponders; Bio Medic Data System, Inc.) and left to recover for 1 wk. Next, mice were either treated with 200 µl of PBS or LPS (4 mg/kg) via i.p. injection. 2 h pt, 10 µl of blood was collected via tail snip into 150 µl of RNALater (Ambion) and frozen at –80°C. RNA isolation and subsequent qRT-PCR was performed as previously described in our Materials and methods. At 6 h pt, mice were injected with a lethal dose of LPS (54 mg/kg) and pathologies were recorded for every 2 h for 20 h (weight and temperature). If the C3aR-inhibitor (C3aRi; SB290157; Santa Cruz Biotechnology, Inc.) was used, it was injected at 30 mg/kg with LPS in PBS.
In mice tracked for temperature and weight, 1 wk before sepsis, mice were anesthetized locally with 1% lidocaine (4 mg/kg) on the right side hind-back using a 0.5 ml syringe. After 15 min, electronic temperature and ID transponders were implanted subcutaneously in the same right side hind-back region (IPTT-300 transponders; Bio Medic Data System, Inc.) and left to recover for 1 wk. Next, mice were either treated with 200 µl of PBS or LPS (4 mg/kg) via i.p. injection. 2 h pt, 10 µl of blood was collected via tail snip into 150 µl of RNALater (Ambion) and frozen at –80°C. RNA isolation and subsequent qRT-PCR was performed as previously described in our Materials and methods. At 6 h pt, mice were injected with a lethal dose of LPS (54 mg/kg) and pathologies were recorded for every 2 h for 20 h (weight and temperature). If the C3aR-inhibitor (C3aRi; SB290157; Santa Cruz Biotechnology, Inc.) was used, it was injected at 30 mg/kg with LPS in PBS.