title: Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis.
abstract: New efforts to understand complex interactions between diet, gut microbiota, and intestinal immunity emphasize the need for a standardized murine protocol that has been optimized for the isolation of lamina propria immune cells. In this study multiple mouse strains including BALB/c, 129S6/Sv/EvTac and ICR mice were utilized to develop an optimal protocol for global analysis of lamina propria leukocytes. Incubation temperature was found to significantly improve epithelial cell removal, while changes in media formulation had minor effects. Tissue weight was an effective method for normalization of solution volumes and incubation times. Collagenase digestion in combination with thermolysin was identified as the optimal method for release of leukocytes from tissues and global immunophenotyping, based on the criteria of minimizing marker cleavage, improving cell viability, and reagent cost. The effects of collagenase in combination with dispase or thermolysin on individual cell surface markers revealed diverse marker specific effects. Aggressive formulations cleaved CD8α, CD138, and B220 from the cell surface, and resulted in relatively higher expression levels of CD3, γδ TCR, CD5, DX5, Ly6C, CD11b, CD11c, MHC-II and CD45. Improved collagenase digestion significantly improved viability and reduced debris formation, eliminating the need for density gradient purification. Finally, we demonstrate that two different digestion protocols yield significant differences in detection of CD4(+) and CD8(+) T cells, NK cells, monocytes and interdigitating DC (iDC) populations, highlighting the importance and impact of cell collection protocols on assay outputs. The optimized protocol described herein will help assure the reproducibility and robustness of global assessment of lamina propria immune responses. Moreover, this technique may be applied to isolation of leukocytes from the entire gastrointestinal tract.
authors: Department of Clinical Sciences, Colorado State University, 300 W. Drake Street, Fort Collins, CO 80523, USA. Electronic address: goody13@colostate.edu.
authors: Andrew W
authors: AW
authors: Goodyear
authors: Department of Clinical Sciences, Colorado State University, 300 W. Drake Street, Fort Collins, CO 80523, USA. Electronic address: Ajay.Kumar@colostate.edu.
authors: Ajay
authors: A
authors: Kumar
authors: Department of Clinical Sciences, Colorado State University, 300 W. Drake Street, Fort Collins, CO 80523, USA. Electronic address: sdow@colostate.edu.
authors: Steven
authors: S
authors: Dow
authors: Department of Clinical Sciences, Colorado State University, 300 W. Drake Street, Fort Collins, CO 80523, USA; Department of Environmental and Radiological Health Sciences, Colorado State University, 200 W. Lake Street, Fort Collins, CO 80523, USA. Electronic address: e.p.ryan@colostate.edu.
authors: Elizabeth P
authors: EP
authors: Ryan
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005260:Female; D016130:Immunophenotyping / Q000379:methods; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D007421:Intestine, Small / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D015703:Antigens, CD
keywords: Collagenase digestion; Flow cytometry; Global analysis; Isolation; Small intestine