Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes
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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.
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Enzymatic digestion causes substantial loss of some cell-surface receptors.
“Of note, we observed substantial loss of some cell-surface receptors following enzymatic digestion, in line with previous work in peripheral immunology (Supplementary Fig. 1d–f and Supplementary Note 1)3,5–9.”
From Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain (2022) · cites this paper as [7]
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Respiratory mononuclear phagocytes during steady state and sarcoidosis
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Antigen-Presenting Cells and Peripheral Neurons in HIV Transmission
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- Authors
- Botting, R. A., Bertram, K. M., Baharlou, H., Sandgren, K. J., Fletcher, J., Rhodes, J. W., Rana, H., Plasto, T. M., Wang, X. M., Lim, J. J. K., Barnouti, L., Kohout, M. P., Papadopoulos, T., Merten, S., Olbourne, N., Cunningham, A. L., Haniffa, M., & Harman, A. N.
- Affiliations
- The University of Sydney (AU)
- Newcastle University (GB)
- Westmead Institute for Medical Research (AU)
- Parramatta , New South Wales , Australia
- Australia Plastic Surgery , Sydney, New South Wales , Australia
- Cosmetic Culture , Pyrmont, New South Wales , Australia
- Pure Aesthetics Plastic Surgery , Sydney, New South Wales , Australia
- Palm Beach , New South Wales , Australia
- Royal Victoria Infirmary (GB)
- Corresponding author
- Harman, Andrew N
- ORCID
- Venue
- Journal of Leukocyte Biology
- Publisher
- Oxford University Press (OUP)
- Published
- 2017-03-07
- Volume
- 101
- Issue
- 6
- Pages
- 1393-1403
- ISSN
- 0741-5400 (ISSN-L)
- 1938-3673
- PMC
- PMC5433859
- OpenAlex
- W2592533921
- Cited by
- 52
- References
- 36
- Citations by year
- 2026: 5 · 2025: 3 · 2024: 2 · 2023: 5 · 2022: 9 · 2021: 11 · 2020: 8 · 2019: 7 · 2018: 1 · 2017: 1
- FWCI
- 2.10
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Leukocyte Biology (publishedVersion, cc-by-nc)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Newcastle University ePrints (Newcastle Univesity) (cc-by)
- Funders
- National Health and Medical Research Council (WT107931/Z/15/Z)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
- Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
- Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
- Keywords
- monocyte isolation, enzymatic digestion, collagenase, spontaneous migration, cDC1, thrombomodulin, culture conditions
- MeSH terms
- Cell Movement, Cell Separation, Enzymes, Humans, Monocytes, Phagocytes, Phenotype, Skin
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