Mouse and human il 10

Cells were seeded and incubated overnight in 24-well plates (J774A.1 cells, 1 × 10⁵ cells/well; human monocytes, 2.5 × 10⁵ cells/well), and treated with KW3110 (1.25–5 μg/mL for J774A.1 cells and 100 μg/mL for human monocytes) for 24 h at 37 ˚C. To measure IL-1β, the cells were treated with 1 μg/mL of IL-10 or primed with 10 μg/mL of LPS for 4 h, and stimulated with 2 mM ATP for 1 h. For the supernatant transferal experiments, in Fig 1B, the supernatants collected from KW3110-treated J774A.1 cells were transferred to other cells which were then primed with 10 μg/mL of LPS for 4 h and stimulated with 2 mM ATP for 1 h. For the phagocytosis blocking experiments, 1 μg/mL of cytochalasin D was added 30 min prior to KW3110 treatment of J774A.1 cells. The supernatants were then collected and centrifuged at 5,000 rpm for 2 min. Cytokine levels were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits (mouse and human IL-10, BD Biosciences, San Jose, CA, USA; mouse IL-1β, eBioscience, San Diego, CA, USA; human IL-1β, R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Cells were seeded and incubated overnight in 24-well plates (J774A.1 cells, 1 × 10⁵ cells/well; human monocytes, 2.5 × 10⁵ cells/well), and then treated with KW3110 or strains A–D (5 μg/mL for J774A.1 cells and 10 μg/mL for human monocytes) for 24 h at 37 °C. For IL-1β measurement, J774A.1 cells were primed with 10 μg/mL of LPS for 4 h, and then stimulated with 2 mM ATP for 1 h. The supernatant was collected and centrifuged at 5000 rpm for 2 min. Cytokine levels were measured with a commercial enzyme-linked immunosorbent assay (ELISA) kit (mouse and human IL-10, BD Biosciences, San Jose, CA, USA; mouse IL-1β, eBioscience, San Diego, CA, USA).