Animal Treatment and cecal ligation and puncture (CLP)
Mice were anesthetized with isofluorane and a midline abdominal incision was made. The cecum was mobilized, ligated below the ileocecal valve, and punctured twice with a 22 gauge needle to induce polymicrobial peritonitis. Sham-operated mice underwent the same procedure, including opening the peritoneum and exposing the bowel, but without ligation and needle perforation of the cecum. After surgery, the mice were injected with 1 mL physiologic saline solution for fluid resuscitation and 20ug butorphanol for post-operative analgesia. All mice had unlimited access to food and water both pre- and post-operatively, allowed to die spontaneously from sepsis. According to the literature and our previous research, 5 mg/kg CD28 functional antibody was administered intraperitoneally at the same time[21]. Mice were scored with murine sepsis severity scale and survival rate by two independent, blinded researchers.