Fig 2: Assessment of neutrophil survival and activation using analysis by flow cytometry. Flow cytometry was performed to assess (1) neutrophil survival using propidium iodide (PI) and Annexin-V staining, and (2) activation using anti-CD66b and anti-CD11b antibodies, in the following conditions (1): Organoid differentiation medium (ODM) (2), Roswell Park Memorial Institute 1640 medium (RPMI) (3), ODM + lipopolysaccharide (LPS), and (4) RPMI + LPS. (A) Neutrophil survival was assessed at during multiple timepoints up until 72 (h) Survival rates decreased steadily until 24 h in all groups with no significant differences between groups. After 24 h, neutrophils in RPMI only showed significantly lower survival rates, suggesting a detrimental effect of RPMI alone on neutrophil viability. At 72 h, increased survival of neutrophils in LPS was observed. (B, D) Neutrophil activation was analyzed by CD66b (B) and CD11b (D) expression for 72 h at multiple timepoints. Activation markers (CD66b and CD11b) increased across all conditions at 24 h, with neutrophils in RPMI only showing similar activation levels to other conditions after 48 (h). This suggests a delayed activation in RPMI compared to other groups. (C, E) CD66b (C) and CD11b (E) signal intensity were measured at multiple time points up to 72 (h) CD66b signal intensity rose in (1) RPMI (2), ODM, and (3) ODM+LPS, while the signal intensity in RPMI+LPS solely rose within the first 12 h, upon which a decrease was seen (C). CD11b intensity increased over time and plateaued after 12 h, with RPMI showing a less pronounced increase, indicating delayed activation (E).

Fig 3: Analyses by flow cytometry of neutrophil–organoid cocultures. (A) NEC and control (CO) organoid apoptosis (Zombie Aqua staining) increased non-significantly across most conditions, with a significant rise observed in NEC organoids upon LPS stimulation. Additionally, significant increases in apoptosis were detected when comparing NEC or CO organoids alone versus their respective LPS + neutrophil cocultures. (B) TLR-4 expression in organoids was significantly elevated in all conditions following LPS stimulation or coculture with LPS-stimulated neutrophils, with the pronounced effect observed in NEC organoids. (C) Neutrophils were cultured for 24 h under six conditions (1): in ODM alone (2), in ODM + LPS (3), with CO organoids (4), with CO organoids + LPS (5), with NEC organoids, and (6) with NEC organoids + LPS. LPS stimulation in monoculture (group 2) significantly increased neutrophil apoptosis compared to the unstimulated control (group 1). In contrast, apoptosis rates were significantly reduced in all coculture conditions (groups 3–6) compared to the LPS-only monoculture, indicating a potential protective effect mediated by epithelial interaction. (D) Neutrophil TLR-4 expression showed a non-significant increase when cocultured with organoids, but was significantly higher in LPS-stimulated cocultures with NEC versus CO organoids. (E) Neutrophil activation, as measured by CD11b signal intensity, increased significantly when neutrophils (+/– LPS) were cocultured with CO organoids compared to neutrophil monocultures (+/– LPS). This difference was also observed when comparing neutrophils cocultured with LPS-stimulated organoids versus LPS-stimulated neutrophils alone. No significant differences were found in neutrophil activation between CO and NEC cocultures (+/– LPS).