Figure 5

GC-MSCs induce the activation of neutrophils through IL-6–IL-6R interaction. (a) Multiplex screening assays for cytokine/chemokine levels in GC-MSCs supernatants by using Human Cytokine/Chemokine Panel 1. (b) IL-6 levels in GC-MSCs and GCN-MSCs supernatants were determined by using ELISA. (c) Total RNA was extracted from GC-MSCs and GCN-MSCs, and real-time PCR was performed to determine the expression of IL-6 mRNA. (d) Neutrophils were incubated with GC-MSCs or GCN-MSCs-derived conditioned medium. Total RNA was extracted from neutrophils and real-time PCR was performed by using human-specific primers for the quantification of IL-8, CCL2, and TNFα. (e) Neutrophils were cultured with GC-MSCs-derived conditioned medium or control medium in the presence or absence of IL-6RA (10 μg/ml) for 18 h. Total RNA was extracted from neutrophils and real-time PCR was performed by using human-specific primers for the quantification of IL-8, CCL2 and TNFα. (f) After coculture with GC-MSCs CM for 12 h in the presence or absence of IL-6RA (10 μg/ml), neutrophils were harvested and seeded with 2 × 10⁴ SGC-7901 cells in the upper compartment of the chamber. After incubation for 12 h, the migrated cells on the lower surface of the membrane were stained with crystal violet after fixation and then counted (g) HUVECs were seeded at 2 × 10⁴ cells/well with conditioned media from GC-MSCs-primed neutrophils in the presence or absence of IL-6RA (10 μg/ml) for 10 h. The formation of tube-like structure by HUVECs were observed under a phase-contrast microscope and photographed at 100 × magnification. Scale bar=200 μm. **P<0.01