title: Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses.
abstract: Studies on immune cells derived from the human intestine are needed to understand the pathogenesis of gastrointestinal diseases and to develop novel treatment strategies. Isolation techniques to extract these immune cells from intestinal tissue are largely based on murine studies and comparative data on isolation from human intestine is scarce. In this study we evaluated cell yield, viability, and surface-molecule expression on mononuclear leukocytes, comparing three techniques to obtain a single immune cell suspension from human intestine; low concentrations of either the enzymes Collagenase D or Liberase TL, and enzyme-free mechanical dissociation with the Medimachine. Both enzymatic isolation techniques provided a higher cell yield than mechanical dissociation. Expression of surface molecules remained intact after Collagenase D treatment, while Liberase TL digestion resulted in a strong decrease in the expression of the CD4 receptor. Taken together, Collagenase D digestion provides the highest yield of mononuclear cells while keeping surface molecule expression intact.
authors.affiliation: Department of Pediatrics, Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands; Department of Experimental Immunology, AMC, UvA, The Netherlands.
authors.forename: R R C E
authors.initials: RRCE
authors.lastname: Schreurs
authors.affiliation: Department of Experimental Immunology, AMC, UvA, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Drewniak
authors.affiliation: Department of Pediatric Surgery, Pediatric Surgical Center, Emma Children's Hospital, AMC & Free University Medical Center Amsterdam, Amsterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Bakx
authors.affiliation: Department of Pediatrics, Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands.
authors.forename: W E
authors.initials: WE
authors.lastname: Corpeleijn
authors.affiliation: Department of Experimental Immunology, AMC, UvA, The Netherlands.
authors.forename: T H B
authors.initials: THB
authors.lastname: Geijtenbeek
authors.affiliation: Department of Pediatrics, Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands.
authors.forename: J B
authors.initials: JB
authors.lastname: van Goudoever
authors.affiliation: Department of Pediatrics, Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam (UvA), Amsterdam, The Netherlands; Department of Experimental Immunology, AMC, UvA, The Netherlands. Electronic address: m.j.bunders@amc.nl.
authors.forename: M J
authors.initials: MJ
authors.lastname: Bunders
mesh_terms: D017364:Collagenases / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology* / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism*
chemical_list: D017364:Collagenases; C106635:Liberase; D013820:Thermolysin
keywords: Human; Intestine; Isolation method; Lymphocytes; Monocytes