Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Abstract

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6-7 h.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression

“…ECs were isolated from freshly dissected colons by dissociation with DTT and EDTA and separation on a Percoll gradient, as previously described (39). Using this protocol, we typically achieve 99% purity of ECs, with contaminating CD45 + hematopoietic cells at levels of 0.4-1.3% (35).…”

Expression of αvβ8 integrin on dendritic cells regulates Th17 cell development and experimental autoimmune encephalomyelitis in mice

“…Lymphocytes were isolated from the colonic lamina propria as previously described with slight modification (30). Briefly, colons were removed, washed with ice-cold PBS, and cut into 2-to 3-cm-long pieces.…”

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

“…Isolation of LPLs and IELs was performed as described previously (49). Briefly, colons were washed with PBS and cut into 1-cm pieces.…”