0.4 μm pore membrane

Seeded SACC were cells in the Transwell lower chamber, and SH‐SY5Y cells were seeded in the upper chamber of the 0.4 μm pore membrane (Corning). For the in vitro PNI co‐culture system, seeded DRG in the lower chamber, and SACC cells were planted in the upper chamber with a 0.4 μm pore membrane (Corning). The co‐culture system was maintained at 37°C with 5% CO₂ in RPMI‐1640 media with 0.1% bovine serum albumin.

To investigate whether cell-to-cell contact is critical for the reciprocal promotion of cytokine or immunoglobulin production during the ILC3-B-cell interaction, Transwell experiments were performed. In brief, sorted ILC3s were added into the upper chamber of a 24-well Transwell plate (6 × 10⁴ cells/well), and sorted autologous B cells were added into the bottom chamber (3 × 10⁵ cells/well) in the presence of 50 ng/mL IL-7, 50 ng/mL IL-23 and 1 μM ODN2006. The chambers were separated with a 0.4 μm pore membrane (Corning, Kennebunk). All experiments were performed in duplicate. After 4 days, the supernatants in both the upper and bottom chambers were harvested and pooled to detect cytokines and immunoglobulins using enzyme-linked immunosorbent assay (ELISA) and cytometric bead array (CBA) assays.