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Doyle CM, Vine EE, Bertram KM, et al. Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue. Front Immunol. 2021;12:727952doi: 10.3389/fimmu.2021.727952.
Doyle, C. M., Vine, E. E., Bertram, K. M., Baharlou, H., Rhodes, J. W., Dervish, S., Gosselink, M. P., Di Re, A., Collins, G. P., Reza, F., Toh, J. W. T., Pathma-Nathan, N., Ahlenstiel, G., Ctercteko, G., Cunningham, A. L., Harman, A. N., & Byrne, S. N. (2021). Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue. Frontiers in immunology, 12727952. https://doi.org/10.3389/fimmu.2021.727952
Doyle, Chloe M, et al. "Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue." Frontiers in immunology vol. 12 (2021): 727952. doi: https://doi.org/10.3389/fimmu.2021.727952
Doyle CM, Vine EE, Bertram KM, Baharlou H, Rhodes JW, Dervish S, Gosselink MP, Di Re A, Collins GP, Reza F, Toh JWT, Pathma-Nathan N, Ahlenstiel G, Ctercteko G, Cunningham AL, Harman AN, Byrne SN. Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue. Front Immunol. 2021 Sep 01;12:727952. doi: 10.3389/fimmu.2021.727952. eCollection 2021. PMID: 34566985; PMCID: PMC8462295.
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Front Immunol. 2021 Sep 01;12:727952. doi: 10.3389/fimmu.2021.727952. eCollection 2021.

Optimal Isolation Protocols for Examining and Interrogating Mononuclear Phagocytes From Human Intestinal Tissue.

Frontiers in immunology

Chloe M Doyle, Erica E Vine, Kirstie M Bertram, Heeva Baharlou, Jake W Rhodes, Suat Dervish, Martijn P Gosselink, Angelina Di Re, Geoffrey P Collins, Faizur Reza, James W T Toh, Nimalan Pathma-Nathan, Golo Ahlenstiel, Grahame Ctercteko, Anthony L Cunningham, Andrew N Harman, Scott N Byrne

Affiliations

  1. Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
  2. Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
  3. School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
  4. Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
  5. Westmead Cytometry, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
  6. Department of Colorectal Surgery, Westmead Hospital, Westmead, NSW, Australia.
  7. Storr Liver Centre, The Westmead Institute for Medical Research, Westmead, NSW, Australia.
  8. Blacktown Clinical School, Western Sydney University, Blacktown, NSW, Australia.
  9. Blacktown Hospital, Western Sydney Local Area Health District (WSLHD), Blacktown, NSW, Australia.

PMID: 34566985 PMCID: PMC8462295 DOI: 10.3389/fimmu.2021.727952

Abstract

The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin

Copyright © 2021 Doyle, Vine, Bertram, Baharlou, Rhodes, Dervish, Gosselink, Di Re, Collins, Reza, Toh, Pathma-Nathan, Ahlenstiel, Ctercteko, Cunningham, Harman and Byrne.

Keywords: dendritic cells (DC); enzymatic digestion; flow cytometry; human tissue; intestine; macrophage – cell; mononuclear phagocyte cells (MNP)

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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