title: Isolation and cytokine analysis of lamina propria lymphocytes from mucosal biopsies of the human colon.
abstract: Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment; hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10010, USA.
authors.forename: Rowann
authors.initials: R
authors.lastname: Bowcutt
authors.affiliation: Department of Medicine, Division of Gastroenterology, New York University School of Medicine, New York, NY, USA.
authors.forename: Lisa B
authors.initials: LB
authors.lastname: Malter
authors.affiliation: Department of Medicine, Division of Gastroenterology, New York University School of Medicine, New York, NY, USA.
authors.forename: Lea Ann
authors.initials: LA
authors.lastname: Chen
authors.affiliation: Department of Medicine, Division of Gastroenterology, New York University School of Medicine, New York, NY, USA.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Wolff
authors.affiliation: Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Robertson
authors.affiliation: Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Rifkin
authors.affiliation: Department of Medicine, Division of Gastroenterology, New York University School of Medicine, New York, NY, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Poles
authors.affiliation: Department of Medicine, Division of Gastroenterology, New York University School of Medicine, New York, NY, USA.
authors.forename: Ilseug
authors.initials: I
authors.lastname: Cho
authors.affiliation: Department of Microbiology, New York University School of Medicine, New York, NY 10010, USA. Electronic address: png.loke@nyumc.org.
authors.forename: P'ng
authors.initials: P
authors.lastname: Loke
mesh_terms: D001706:Biopsy; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D003093:Colitis, Ulcerative / Q000276:immunology*; D003106:Colon / Q000166:cytology / Q000276:immunology; D003113:Colonoscopy; D003424:Crohn Disease / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism*; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000276:immunology; D019394:Ki-67 Antigen / Q000378:metabolism; D064307:Microbiota / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D016207:Cytokines; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D019394:Ki-67 Antigen; D016212:Transforming Growth Factor beta
keywords: Biopsies; Cytokines; Flow cytometry; Lamina propria mononuclear cells