Although the inflammatory cytokine IL-10 is pivotal in regulatory B-cell function, detecting IL-10-producing B cells by intracellular IL-10 staining requires multiple steps and tedious preparation. In contrast, the Il10-eGFP reporter mouse model (VertX), generated in 2009, allows easier and quicker detection of IL-10-producing B cells with the possibility of sorting viable cells without membrane permeabilization and ex vivo activation. Even though detecting IL-10+ cells is simpler, several nuances are important. For example, methanol-containing buffers delete GFP signal, while long-term fixation can maintain GFP intensity but decreases other intracellular signals (FOXP3, etc.). Here, we provide optimized and improved protocols for GFP detection in intestinal B cells and isolation techniques of lamina propria, spleen, mesenteric lymph node, peritoneum, and blood cells from VertX mice.
Keywords: Flow cytometry; GFP; Gnotobiotic mice; Interleukin-10; Microbiota; Mucosal immunology; Regulatory B cell.