title: Isolation of viable and functional T-cells from human palatine tonsils.
abstract: Increasing clinical evidence indicates that removal of the palatine tonsils enhances the risk for adults to suffer from severe illnesses. Together with recent experimental findings pointing to the presence of immunologically competent immune cells these findings illustrate that adult palatine tonsils likely play an appreciable role in the host immune response. T-cells are abundant in the palatine tonsil and are a pivotal entity of the adaptive immune response. However, investigation of T-cells from tonsils has been widely neglected and largely restricted to immune phenotyping. Accordingly, methodological literature describing the experimental preparation and isolation of T-cells from tonsils is scarce and has rarely been complemented with rigorous tests of T-cell functionality. We report here on a comparative investigation of three isolation protocols composed of permutations of different tissue grinding approaches, density gradient centrifugation and automated magnetic collection of CD4/CD8 T-cells. Importantly we put a strong emphasis on assessing the impact of the preparative procedures on the functionality of T-cells at the level of viability and functional response to T-cell receptor (TCR) ligation. The reported, optimized preparation protocols allow for the rapid isolation of highly viable, functional T-cells within 2.5h and represent a useful, affordable approach for the analysis of tonsillar T-cells.
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Weigel
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany; Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Geißler
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany.
authors.forename: Robby
authors.initials: R
authors.lastname: Markwart
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Schubert
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany; Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rubio
authors.affiliation: Department of Otorhinolaryngology, Jena University Hospital, Jena, Germany. Electronic address: Orlando.Guntinas@med.uni-jena.de.
authors.forename: Orlando
authors.initials: O
authors.lastname: Guntinas-Lichius
authors.affiliation: Integrated Research and Treatment Center for Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany. Electronic address: Robert.Requardt@med.uni-jena.de.
authors.forename: Robert Pascal
authors.initials: RP
authors.lastname: Requardt
mesh_terms: D000328:Adult; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D014066:Palatine Tonsil / Q000166:cytology* / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D055815:Young Adult
keywords: Palatine tonsil; Purification of T-cells; T-cell activation; Tonsillitis