Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Abstract

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

L13a-dependent translational control in macrophages limits the pathogenesis of colitis

“…[21][22][23] Pieces (1.5 cm) of intestinal tissue were digested from DSS-water-fed and normal drinking water-fed animals and subjected to Type VIII collagenase and DNase I (Sigma-Aldrich) digestion through orbital shaking at 200 rpm for 20 minutes at 37uC. Occasional vortexing was performed to ensure the complete dissociation of the intestinal tissue.…”

Oral Norovirus Infection Is Blocked in Mice Lacking Peyer's Patches and Mature M Cells

“…Small intestines were harvested from naïve WT and Rag-␥c Ϫ/Ϫ BALB/c mice and were digested with 1.5 mg/ml type VIII collagenase (Sigma-Aldrich) and 40 g/ml DNase I (Roche) in order to collect the lamina propria mononuclear cells, as described previously (33). For flow cytometry immunophenotyping experiments, 3 ϫ 10 6 cells per well were surface stained for 30 min on ice in calcium-and magnesium-free Hanks balanced salt solution (HBSS) with 5% fetal bovine serum (FBS).…”

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

“…Make a small incision in skin of the abdomen with scissors and then make an incision to expose the peritoneal cavity. Similar procedures can be found described here 12 . 3.…”