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  • Ravindran, AvinashKarolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Med Solna, Immunol & Allergy Unit, Stockholm, Sweden (författare)

An Optimized Protocol for the Isolation and Functional Analysis of Human Lung Mast Cells

  • Artikel/kapitelEngelska2018

Förlag, utgivningsår, omfång ...

  • 2018-10-05
  • FRONTIERS MEDIA SA,2018
  • electronicrdacarrier

Nummerbeteckningar

  • LIBRIS-ID:oai:DiVA.org:uu-367035
  • https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-367035URI
  • https://doi.org/10.3389/fimmu.2018.02193DOI
  • http://kipublications.ki.se/Default.aspx?queryparsed=id:139270221URI
  • https://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-152055URI

Kompletterande språkuppgifter

  • Språk:engelska
  • Sammanfattning på:engelska

Ingår i deldatabas

Klassifikation

  • Ämneskategori:ref swepub-contenttype
  • Ämneskategori:art swepub-publicationtype

Anmärkningar

  • Funding Agencies|Swedish Research Council-Medicine and Health; Heart-Lung Foundation; Ollie and Elof Ericssons foundation; Ellen; Walter and Lennart Hesselmans foundation; European Cooperation in Science and Technology COST Action, Developmental Origins of Chronic Lung Disease [BM1201]; ChAMP (Centre for Allergy Research Highlights Asthma Markers of Phenotype) consortium - Swedish Foundation for Strategic Research; Karolinska Institutet; AstraZeneca & Science for Life Laboratory Joint Research Collaboration; Vardal Foundation
  • Background: Mast cells are tissue-resident inflammatory cells defined by their high granularity and surface expression of the high-affinity IgE receptor, Fc + RI, and CD117/KIT, the receptor for stem cell factor (SCF). There is a considerable heterogeneity among mast cells, both phenotypically and functionally. Human mast cells are generally divided into two main subtypes based on their protease content; the mucosa-associated MCT (tryptase positive and chymase negative mast cell) and the connective tissue associated-residing MCTC (tryptase and chymase positive mast cell). Human lung mast cells exhibit heterogeneity in terms of cellular size, expression of cell surface receptors, and secreted mediators. However, knowledge about human lung mast cell heterogeneity is restricted to studies using immunohistochemistry or purified mast cells. Whereas the former is limited by the number of cellular markers that can be analyzed simultaneously, the latter suffers from issues related to cell yield.Aim: To develop a protocol that enables isolation of human lung mast cells at high yields for analysis of functional properties and detailed analysis using single-cell based analyses of protein (flow cytometry) or RNA (RNA-sequencing) expression.Methods: Mast cells were isolated from human lung tissue by a sequential combination of washing, enzymatic digestion, mechanical disruption, and density centrifugation using Percoll (WEMP). As a comparison, we also isolated mast cells using a conventional enzyme-based protocol. The isolated cells were analyzed by flow cytometry.Results: We observed a significant increase in the yield of total human lung CD45(+) immune cells and an even more pronounced increase in the yield of CD117(+) mast cells with the WEMP protocol in comparison to the conventional protocols. In contrast, the frequency of the rare lymphocyte subset innate lymphoid cells group 2 (ILC2) did not differ between the two methods.Conclusion: The described WEMP protocol results in a significant increase in the yield of human lung mast cells compared to a conventional protocol. Additionally, the WEMP protocol enables simultaneous isolation of different immune cell populations such as lymphocytes, monocytes, and granulocytes while retaining their surface marker expression that can be used for advanced single-cell analyses including multi-color flow cytometry and RNA-sequencing.

Ämnesord och genrebeteckningar

Biuppslag (personer, institutioner, konferenser, titlar ...)

  • Rönnberg, ElinKarolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Med Solna, Immunol & Allergy Unit, Stockholm, Sweden (författare)
  • Dahlin, Joakim S.Karolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Med Solna, Immunol & Allergy Unit, Stockholm, Sweden (författare)
  • Mazzurana, LucaKarolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Med Huddinge, Ctr Infect Med, Stockholm, Sweden (författare)
  • Säfholm, JesperKarolinska Inst, Inst Environm Med, Unit Expt Asthma & Allergy Res, Ctr Allergy Res, Stockholm, Sweden (författare)
  • Orre, Ann-CharlotteKarolinska Univ Hosp, Karolinska Inst, Dept Mol Med & Surg, Thorac Surg, Stockholm, Sweden (författare)
  • Al-Ameri, MamdohKarolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Mol Med & Surg, Thorac Surg, Stockholm, Sweden (författare)
  • Peachell, PeterUniv Sheffield, Royal Hallamshire Hosp, Acad Unit Resp Med, Sheffield, S Yorkshire, England (författare)
  • Adner, MikaelKarolinska Institutet,Karolinska Inst, Inst Environm Med, Unit Expt Asthma & Allergy Res, Ctr Allergy Res, Stockholm, Sweden (författare)
  • Dahlen, Sven-ErikKarolinska Institutet,Karolinska Inst, Inst Environm Med, Unit Expt Asthma & Allergy Res, Ctr Allergy Res, Stockholm, Sweden (författare)
  • Mjösberg, JennyLinköpings universitet,Karolinska Institutet,Karolinska Univ Hosp, Karolinska Inst, Dept Med Huddinge, Ctr Infect Med, Stockholm, Sweden;Linkoping Univ, Dept Clin & Expt Med, Linkoping, Sweden,Avdelningen för Kirurgi, Ortopedi och Onkologi,Medicinska fakulteten(Swepub:liu)jenmj65 (författare)
  • Nilsson, GunnarKarolinska Institutet,Uppsala universitet,Hematologi,Karolinska Univ Hosp, Karolinska Inst, Dept Med Solna, Immunol & Allergy Unit, Stockholm, Sweden,Karolinska Univ Hosp, Sweden; Uppsala Univ, Sweden(Swepub:uu)gunnarns (författare)
  • Karolinska InstitutetKarolinska Univ Hosp, Karolinska Inst, Dept Med Solna, Immunol & Allergy Unit, Stockholm, Sweden (creator_code:org_t)

Sammanhörande titlar

  • Ingår i:Frontiers in Immunology: FRONTIERS MEDIA SA91664-3224

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