title: Isolation and high-dimensional phenotyping of gastrointestinal immune cells.
abstract: The gastrointestinal immune system plays a pivotal role in the host relationship with food antigens, the homeostatic microbiome and enteric pathogens. Here, we describe how to collect and process liver and intestinal samples to efficiently isolate and analyse resident immune cells. Furthermore, we describe a step-by-step methodology showing how to high-dimensionally immunophenotype resident leucocytes using cytometry by time-of-flight, providing a well-characterized antibody platform that allows the identification of every leucocyte subset simultaneously. This protocol also includes instructions to purify and cultivate primary murine hepatocytes, a powerful tool to assess basic cell biology and toxicology assays. Gut and liver samples from the same mouse can be collected, processed and stained in less than 6 hr. This protocol enables the recovery of several populations of purified and viable immune cells from solid and fibrous organs, preventing unwanted loss of adherent cells during isolation.
authors.affiliation: Departamento de Morfologia, Center for Gastrointestinal Biology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Bruna A
authors.initials: BA
authors.lastname: David
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Rubino
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Thais G
authors.initials: TG
authors.lastname: Moreira
authors.affiliation: Departamento de Morfologia, Center for Gastrointestinal Biology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: Freitas-Lopes
authors.affiliation: Departamento de Morfologia, Center for Gastrointestinal Biology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Araújo
authors.affiliation: Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Paul
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
authors.forename: Rafael M
authors.initials: RM
authors.lastname: Rezende
authors.affiliation: Departamento de Morfologia, Center for Gastrointestinal Biology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Gustavo B
authors.identifier: 0000-0001-9887-3616
authors.initials: GB
authors.lastname: Menezes
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry; D016130:Immunophenotyping / Q000379:methods*; D007413:Intestinal Mucosa / Q000166:cytology* / Q000276:immunology; D007962:Leukocytes / Q000166:cytology*; D008099:Liver / Q000166:cytology* / Q000276:immunology; D008198:Lymph Nodes / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL
keywords: cell isolation; cytometry by time of flight; lamina propria; liver; mass cytometry