Discontinuous percoll

Gastric epithelial cells were isolated and prepared according to a modification of a previously published method [22 (link)]. Dissected small segments of the stomach were agitated at room temperature for 10 min in a Hank's balanced salt solution (HBSS) (Thermo Fisher Scientific Inc., Waltham, MA) medium containing 1 mM DTT. After removal of the supernatant, the tissues were stirred at 37 °C for 10 min in HBSS containing 10 mM EDTA. After removal of the supernatant, the tissue suspension was passed through a nylon mesh to remove debris and centrifuged through a 25/40 % discontinuous Percoll (Sigma-Aldrich, St. Louis, MO) gradient at 600 × g at 20 °C for 20 min. The cells collected from the interface of 25/40 % were the epithelial cells. Lysates were prepared from tissues and cells and analyzed by western blotting, according to a previously published method [23 (link)]. The Abs used in western blotting are summarized in Additional file 1: Table S1.

The preparation of primary O. niloticus hepatocytes was performed as in previous descriptions (31 (link), 35 (link)–37 (link)). Briefly, the livers were dissected and digested with 0.1% trypsin (0.2% EDTA) (Sigma, USA) for 20 min. After washing, the cells were re-suspended with the addition of a 3-mL red blood cell lysis buffer (KangWei, China) on the ice for 3 min. Then the cells were washed and adjusted to 1 × 10⁶ cells/mL within the DMEM medium.
Monocytes/macrophages (MO/MΦ) were separated from the head kidney as in previous descriptions (33 (link), 37 (link)–39 (link)). Briefly, the cells were isolated through a 54%/31% discontinuous percoll (Sigma, USA) density gradient and centrifuged at 500 × g at 4°C for 40 min. The cells were collected and adjusted to 1 × 10⁷ cells/mL with the L-15 medium (Gibico, USA), then finally cultured at least 24 h at 25°C. After removing the non-adherent cells, the MO/MΦ were re-suspended and adjusted to 1 × 10⁶ cells/mL with the L-15 medium.
The experimental group was stimulated with formalin-inactivated S. agalactiae or A. hydrophila (1 × 10⁷ CFU/mL), and the control group was only stimulated with sterile 1 × PBS. All the groups were maintained at 25°C, and cells were collected at the time points of 0, 3, 6, 12, 24, and 48 h post-stimulation.

Lamina propria lymphocytes (LPLs) from the jejunum were isolated as reported by us earlier [28 (link),30 (link)]. In brief, jejunum biopsies were obtained by endoscopy, washed properly in chilled sterile PBS, minced with scissors, and digested with EDTA solution in a 37 °C shaker for 30 min to detach the majority of intestinal epithelial cells. After the removal of epithelial cells by filtration through screen cup strainer with 50 mesh size (0.229 mm) (Sigma-Aldrich, St. Louis, MO, USA), the remaining tissues on the strainer were scrapped and further minced with scissors and digested with media containing 60 U/mL of collagenase (type II, Sigma-Aldrich). The cell suspension was passed through a 16-gauge feeding needle for better separation of any clumps. The larger clumps were removed using a nylon biopsy bag (ThermoFisher, Waltham, MA, USA). To enrich LPLs, cells were centrifuged over discontinuous percoll (Sigma-Aldrich) gradients. The interface layer containing LPLs between the two percoll layers was collected, washed in PBS, and resuspended with complete media. This percoll separation removed dead cells and enriched the cell population for lymphocytes. However, these enriched LPLs also contain trace amount of epithelial and other leukocyte positive cells as reported by us elsewhere [31 (link)]. The enriched LPLs were used for flow cytometry assays.