Peritoneal macrophages were prepared as described previously96. Briefly, 2 ml of 4% thioglycollate (Difco #225640) solution was intraperitoneally injected into mice. Five days after the injection, peritoneal cells were collected by washing the peritoneum cavity with 5 ml of RPMI 1640 medium containing 10% FCS and 2-mercaptoethanol (RPMI-10). The collected cells were cultured overnight in RPMI-10 and then the adherent cells were used for assays. BMDMs were prepared as described previously27. Briefly, bone marrow cells collected from WT, Trem2−/−, Tyrobp−/−, or Clex4e−/− mice were cultured in RPMI-10 in the presence of 25 ng of recombinant murine M-CSF (PeproTech #AF-315-02) for 3 days, and then the adherent cells were collected as BMDMs. For in vitro cell stimulation, the lipid solutions were added into the 96-well flat bottom plates at 20 μl/well and then the solvent was completely evaporated in a hood before plating macrophages30. A total of 1 × 105 cells/well were stimulated with plate-coated lipids or TLR ligands in RPMI-10. The culture supernatants after 24-h culture were collected and the concentrations of TNF (eBioscience #88-7324-88), IL-6 (eBioscience #88-7024-88), IL-12p40 (Biolegend #431604), IL-10 (Biolegend #431411), and MCP-1 (Biolegend #432701) were analyzed by ELISA kits, according to manufacturer’s instructions. For Syk inhibition, cells were incubated with 1 μM BAY-613606 (Calbiochem #574714) for 30 min prior to the stimulation.

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