L2280

In this study, gram-negative sepsis-induced cardiomyopathy model was established with lipopolysaccharide (LPS) (Sigma; L2280) or cecal ligation and puncture (CLP) [52 (link), 53 (link)]. For LPS-induced cardiomyopathy, mice weighed 22.3 ± 2.5 g were randomly underwent an intraperitoneal injection of LPS (10 mg/kg) or saline equally. The general appearance and heart function of the animals were monitored. After LPS stimulation for 24 h, the mice were sacrificed for subsequent experiments. The CLP surgery procedure was similar to previous studies with minor modifications [52 (link)]. Briefly, mice were anesthetized with 2% isoflurane (RWD, R510-22-4) with a maintained 37 °C body temperature. After exposure through a small anterior abdominal incision, the cecum was ligated with a 4–0 silk suture and was punctured twice with an 18-gauge needle. The cecum was then gently squeezed to extrude a small amount of stool from the puncture site to ensure complete perforation. Next, the cecum was relocated into the peritoneal cavity, and the abdominal incision was closed with 6–0 silk sutures. Sham-operated mice underwent the same operation procedure but without CLP. Subsequent experiments were performed 24 h after the operation.

Immortalized bone marrow-derived macrophages (iBMMs) and iBMMs expressing ASC-citrine were developed in the laboratory of Jonathan Kagan through retroviral transduction of primary murine bone marrow-derived macrophages from WT mice or transgenic mice expressing ubiquitous CRE on a WT ASC background (transgenic mice developed in the Golenbock lab) [18 (link),20 (link)] and were a kind gift from Jonathan Kagan and Charles Evavold. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Heat-Inactivated Fetal Bovine Serum (FBS), penicillin, streptomycin, L-glutamine, sodium pyruvate, and β-mercaptoethanol. Cells were washed in phosphate buffered saline (PBS) pH 7.4 containing 2 mM EDTA to detach cells for passage. Cells were passaged 1:10 every 3 days. Lipopolysaccharide (LPS) from E. coli serotype O55:B5 (L2280, Sigma Aldrich, St. Louis, MO, USA) was reconstituted at 1 mg/mL, aliquoted, and frozen at −80 °C for later use. The 5′adenosine triphosphate (ATP, A6419, Sigma Aldrich) was resuspended at 500 mM, adjusted to pH 7.4, and stored at −20 °C. Propidium iodide (PI, P4864, Sigma Aldrich) was purchased in a ready-to-use stock solution at a concentration of 1 mg/mL.