Operating processes for preparing cell suspension from zebrafish intestinal mucosa. (A) Schematic diagram of operating processes. (B) Photographs of the detail operation, including dissection, intestinal separation, blowing and filtering.

Quality control of prepared intestinal single cell suspension from Tg(lyz:DsRED2);Tg(mpeg1:EGFP). (A) Imaging by automated cell counter, cell under brightfield and fluorescence channels (FL1: 488nm; FL2:558nm). Scale bar: 100μm. The cellular diameter of the prepared intestinal mucosal cell suspension was calculated besides. (B) Cell viability reflected AO/PI staining during cytometric analysis. The stained cell number was counted by automatic cell counter. After AO/PI staining, 75% cell obtained from robbing method (RM) was alive, meanwhile for the blowing method, 80% of all cells (AC) and 90% of genetically fluorescence labeled immune cells, which were the target cells (TC), were alive for flow cytometric analysis. (C) Images of intestinal mucosal cells from Tg(lyz:DsRED2);Tg(mpeg1:EGFP). In single cell suspensions, DsRed labeled lyz⁺ cell and EGFP labeled mpeg1⁺ cells could be clearly observed, together with other unstained cells (shown in the brightfield). ** represented p < 0.01, **** represented p < 0.0001.

Enriching intestinal immune cells via stripping the muscular layer. (A) Morphological analysis of HE staining in comparison between intestinal tissue after blowing treatment and normal intestinal tissue. (B) qPCR analysis of genes involved in immune cell differentiation. All values are means ± SEM; statistical significance was determined by independent-samples T-test. For the comparison of IMC (intestinal mucosa cells) vs IT (intestinal tissue), *** represented p < 0.01.

Enriched KEGG pathways and GO terms as well as heatmaps of mucosal immune related DEGs. Intestinal pathways (A) and terms (B) for the comparison between IMC and IT. (C) The heat map of immune cells maker genes. (D) The heat map of mucus related genes. (E) The heat map of mast cell related genes.

Visualization of involved genes in innate immune related KEGG pathways by cnetplot analysis. The pattern recognition related “C-type lectin receptor signaling pathway”, “NOD like receptor signaling pathway”, and “Toll like receptor signaling pathway”, as well as cytokine signaling related “cytokine-cytokine receptor interaction” were shown in details.

Flow cytometric analysis of transgenic zebrafish, including Tg(lyz:DsRED2), Tg(rag2:DsRed), Tg(lck:EGFP), and Tg(mpeg1:EGFP). (A) The gating strategies in cytometric analysis for intestinal neutrophils (with red fluorescence labeled Lyz), immature lymphocytes (with red fluorescence labeled Rag2), mature T lymphocytes (with green fluorescence labeled Lck), and macrophages (with green fluorescence labeled Mpeg1) in zebrafish. Compare with wild type samples, all types of fluorescence labeled immune cells could be accurately sorted. (B) Comparing samples made by mechanical dissociation of whole intestine and blowing off mucosal cells from muscularis, the fluorescence labeled cells were significantly enriched. * represented p < 0.05, **** represented p < 0.0001.

Proportion of fluorescent labeled immune cells, including neutrophils, macrophages, lymphocytes, and activated T cells, in immune organs (the periphery blood, intestine, liver, kidney and spleen). * represented p < 0.05, ** represented 0.01< p < 0.05. and *** represented p < 0.01.

The SBMIE induced mucosa pathology was related to the altered composition of immune cells. The single cell suspension, obtained using current optimized method, accurately reflects altered composition of intestinal immune cells upon soybean induced inflammation. (A) Validation of inflammation by pathological analysis of intestinal mucosa. The length of intestinal villi was reduced at the condition of SBM (soybean meal) feeding. (B) qPCR analysis of genes involved in intestinal inflammation. (C) the increased proportion of intestinal mpeg⁺ and lyz⁺ cells upon SBMIE. *** represented p < 0.01. ns, no significance.