title: Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.
abstract: Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue.
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Botting
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Kirstie M
authors.initials: KM
authors.lastname: Bertram
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Heeva
authors.initials: H
authors.lastname: Baharlou
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Kerrie J
authors.initials: KJ
authors.lastname: Sandgren
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Fletcher
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Jake W
authors.initials: JW
authors.lastname: Rhodes
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Hafsa
authors.initials: H
authors.lastname: Rana
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Toby M
authors.initials: TM
authors.lastname: Plasto
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.
authors.forename: Xin Maggie
authors.initials: XM
authors.lastname: Wang
authors.affiliation: Parramatta, New South Wales, Australia.
authors.forename: Jake J K
authors.initials: JJK
authors.lastname: Lim
authors.affiliation: Australia Plastic Surgery, Sydney, New South Wales, Australia.
authors.forename: Laith
authors.initials: L
authors.lastname: Barnouti
authors.affiliation: Australia Plastic Surgery, Sydney, New South Wales, Australia.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Kohout
authors.affiliation: Cosmetic Culture, Pyrmont, New South Wales, Australia.
authors.forename: Tim
authors.initials: T
authors.lastname: Papadopoulos
authors.affiliation: Pure Aesthetics Plastic Surgery, Sydney, New South Wales, Australia.
authors.forename: Steve
authors.initials: S
authors.lastname: Merten
authors.affiliation: Palm Beach, New South Wales, Australia; and.
authors.forename: Norman
authors.initials: N
authors.lastname: Olbourne
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Anthony L
authors.initials: AL
authors.lastname: Cunningham
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.|Department of Dermatology, Royal Victoria Infirmary, Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom.
authors.forename: Muzlifah
authors.initials: M
authors.lastname: Haniffa
authors.affiliation: Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia; andrew.harman@sydney.edu.au.|The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Harman
mesh_terms: D002465:Cell Movement; D002469:Cell Separation / Q000379:methods*; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D009000:Monocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D010586:Phagocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D010641:Phenotype; D012867:Skin / Q000166:cytology* / Q000276:immunology / Q000378:metabolism
chemical_list: D004798:Enzymes
keywords: DC; ex vivo; human; macrophage