Primary bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland; 12–604 F) plus fetal bovine serum (10% v/v; Gibco, Waltham, MA, USA; 16000–044) and penicillin‒streptomycin-amphotericin B (Lonza; 17–745E). Bone marrow cells were cultured for 5–7 days in DMEM containing 25 ng/mL macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA; 416-ML) to generate fully differentiated BMDMs.
PMs were prepared as previously described36 (link). Briefly, 3 days after IP injection of 3% thioglycolate, mice were euthanized and IP injected with ice-cold phosphate buffered saline (PBS) containing 3% fetal bovine serum (5 mL). The wash solution was collected and centrifuged (1500 rpm, 8 min, 4 °C) to obtain a macrophage pellet. Cells were suspended in culture medium, counted, and seeded in plates for further analysis.
Before experiments, cells were incubated overnight in resting medium (DMEM with 5% fetal bovine serum). Cells were pretreated with vehicle, ATB1021, YTK-2205 or ATB1071 for 1 h and then stimulated with 100 ng/mL LPS (InvivoGen, San Diego, CA; tlrl-eblps), 20 μg/mL poly(I:C) (Sigma‒Aldrich), 10 μg/mL zymosan (Invivogen; tlrl; zyn), or 100 ng/mL Pam3CSK4 (InvivoGen; tlrl-pms) for 3, 6, or 18 h. Supernatants were collected for ELISAs; cells were lysed for RNA preparation or Western blotting. For autophagic flux analysis, bafilomycin A1 (BafA1) (Sigma‒Aldrich, b1793) was preincubated with the cells for 1.5 h at a final concentration of 100 nM.
PMs were prepared as previously described36 (link). Briefly, 3 days after IP injection of 3% thioglycolate, mice were euthanized and IP injected with ice-cold phosphate buffered saline (PBS) containing 3% fetal bovine serum (5 mL). The wash solution was collected and centrifuged (1500 rpm, 8 min, 4 °C) to obtain a macrophage pellet. Cells were suspended in culture medium, counted, and seeded in plates for further analysis.
Before experiments, cells were incubated overnight in resting medium (DMEM with 5% fetal bovine serum). Cells were pretreated with vehicle, ATB1021, YTK-2205 or ATB1071 for 1 h and then stimulated with 100 ng/mL LPS (InvivoGen, San Diego, CA; tlrl-eblps), 20 μg/mL poly(I:C) (Sigma‒Aldrich), 10 μg/mL zymosan (Invivogen; tlrl; zyn), or 100 ng/mL Pam3CSK4 (InvivoGen; tlrl-pms) for 3, 6, or 18 h. Supernatants were collected for ELISAs; cells were lysed for RNA preparation or Western blotting. For autophagic flux analysis, bafilomycin A1 (BafA1) (Sigma‒Aldrich, b1793) was preincubated with the cells for 1.5 h at a final concentration of 100 nM.
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