Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue.
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.
Weigmann
Benno
B
I. Med. Clinic, Immunological Laboratory, Johannes-Gutenberg University, Obere Zahlbacherstrasse 63, 55131 Mainz, Germany. bweigm@uni-mainz.de
Tubbe
Ingrid
I
Seidel
Daniel
D
Nicolaev
Alex
A
Becker
Christoph
C
Neurath
Markus F
MF
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D003106:Colon / Q000166:cytology / Q000276:immunology*; D016616:Cryoultramicrotomy; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology*; D008214:Lymphocytes / Q000166:cytology*; D051379:Mice; D046509:Tissue Culture Techniques