Cell culture and stimulation
To prepare bone marrow derived macrophages (BMDMs), marrow was flushed
out from femurs and tibiae of hind legs using complete RPMI medium
(Invitrogen, Carlsbad, CA, USA). After red blood cells were lysed, cells
were seeded onto cell culture plates in complete RPMI medium with 20
ng/ml M-CSF (R&D Systems, Minneapolis, MN, USA) and cultured for 6
days. To prepare monocytes from human peripheral blood mononuclear
cells, whole blood was layered on top of Histopaque 1077 (Sigma-Aldrich,
St. Louis, MO, USA) and centrifuged at 400 x g for 30 mins at room
temperature. The interface containing mononuclear cells was collected.
Monocytes were then selected using a MagniSort Human Monocyte Enrichment
Kit (Invitrogen, Carlsbad, CA, USA). Cells were stimulated with 200
ng/ml LPS for 4 hours and then treated with 10 or 100 μM LMT503 for 18
hours at 37°C. For IL-1β, BMDMs were stimulated with 200 ng/ml LPS for 3
hours followed by 5 mM ATP (Sigma-Aldrich, St. Louis, MO, USA) for 1
hour, and then treated with 10 or 100 μM LMT503 for 18 hours at 37°C.
For monocytes derived from human peripheral blood mononuclear cells,
fresh blood was collected from healthy adult volunteers from Ajou
University Hospital. This study was approved by the Human Research
Protection Center of Ajou University Hospital (IRB #2022-0534-001).