Male dahl-S rats (S), male C57BL/6J mice, or male Tlr2 knockout mice (B6.129-Tlr2tm1Kir/J; Jackson Laboratory, Bar Harbor, ME, USA) were injected intraperitoneally with thioglycolate, as previously described [31 (link)]. After 72 h, peritoneal macrophages were obtained by lavage and adherent macrophages were allowed to settle for another 72 h. Cells were allowed to grow in 12-well plates and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Catalog No. 11995065; ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Rocky Mountain Biologicals, Inc, Missoula, MT, USA) and 1% penicillin–streptomycin solution (Caisson Labs, Smithfield, UT, USA).
MC-LR was used at a dose of 10 μM. Anti-Tlr2 monoclonal Ab (Item No. mabg-mtlr2; InvivoGen, San Diego, CA, USA), used at a dose of 2.5 μg/mL, with pretreatments for 1 h. Doramapimod (Item No. 10460; Cayman Chemical, Ann Arbor, MI, USA), used at a dose of 10 μM, with pretreatments for 30 min. All treatments were for 24 h in duration. Treatments were preceded by 24 h of serum starvation using DMEM supplemented with only 1% penicillin–streptomycin solution, and treatments were prepared in the same serum-starved conditions.
MC-LR was used at a dose of 10 μM. Anti-Tlr2 monoclonal Ab (Item No. mabg-mtlr2; InvivoGen, San Diego, CA, USA), used at a dose of 2.5 μg/mL, with pretreatments for 1 h. Doramapimod (Item No. 10460; Cayman Chemical, Ann Arbor, MI, USA), used at a dose of 10 μM, with pretreatments for 30 min. All treatments were for 24 h in duration. Treatments were preceded by 24 h of serum starvation using DMEM supplemented with only 1% penicillin–streptomycin solution, and treatments were prepared in the same serum-starved conditions.
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