Thioglycollate

Bone marrow transplantation was performed, as described. Mice were put on a HCD for 10 weeks and subsequently injected with thioglycollate medium (3%, Fischer). Four days after injection, mice were sacrificed and the peritoneum was flushed twice with 10 ml PBS to collect peritoneal macrophages. Flushed thioglycollate-elicited macrophages were cultured in RPMI-1640 25 mM HEPES, 2 mM l-glutamine, 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml) (all Gibco) and allowed to adhere for 3 h. Oil Red O staining (0.3% in 60% isopropanol, Sigma) was performed to determine lipid accumulation. For gene expression experiments, macrophages adhered overnight.

The HEK293T was from American Type Culture Collection, and the iBMDM cell line was a gift from Dr. Feng Shao (National Institute of Biological Sciences, Beijing, China). Peritoneal macrophages were separated by abdomen irrigation with Dulbecco’s Modified Eagle Medium (DMEM; Gibco) from mice 4 days after thioglycollate injection. Cells were plated into cell culture materials and cultured for at least 12 h before use. All cells were cultured in endotoxin-free DMEM (GIBCO) or RPMI1640 (GIBCO), supplemented with 10% FCS (Gicbo). Sendai virus (SeV, RLRs or TLR7/8 ligand) was propagated in 8-10-day-old embryonated chicken eggs. VSV (Indiana Strain, RLRs or TLR7/8 ligand) was propagated and amplified by infection of a monolayer of HEK293T cells. 48 h after infection, the supernatant was harvested and clarified by centrifugation. Viral titer was determined by TCID50 on HEK293T cells. Peritoneal macrophages were harvested from mice 4 days after the injection of the thioglycollate (BD, Sparks, MD). Cells were plated into 12-well plates and cultured in the absence or presence of LPS (100 ng/ml), CpG ODN (0.3 μM), poly(I:C) (10 μg/ml). Cells were infected with VSV (10:1 M.O.I) and SeV (10:1 M.O.I.) for the indicated time. According to the manufacturer’s instructions, cytokine production was analyzed using mouse IL-6 ELISA kits (Thermo).

BALB/c and Slamf1^-/- 8–12 weeks old female mice were injected intraperitoneal with 10% thioglycollate (Gibco, Grand Island, NY) (1 ml per mouse). After 4 days, mice were euthanized by CO₂ inhalation. Peritoneal cells collected by lavage with 10 ml of cold PBS were pelleted by centrifugation (260 g, for 10 min, at 4°C) and seeded in 6 well multi-well plates (10⁶ cells/well) in RPMI medium supplement with 5% FBS overnight at 37°C. Non-adherent cells were removed by gently washing three times with warm PBS. Peritoneal macrophages were infected with trypomastigotes of T. cruzi at a ratio of 5 parasites per cell. At 1, 6 and 24 hours post-infection (hpi), the cells were washed three times with PBS to remove the unbound parasite.