title: A method for extracting tissue proteins for use in lymphocyte function assays.
abstract: Currently human T-cell responses to unpurified tissue extracts cannot be easily measured because current cell lysis methods yield a lysate that is toxic. Here we describe the optimization of a protocol for extracting proteins from human tissues in a format that is compatible with the functional analysis of human T cells. The tissue was homogenized in a mixture of butan-1-ol, acetonitrile and water and then lyophilized. Lyophilized protein extracts were dissolved in 8M urea because urea did not affect T-cell function when present at 0.08 M or less. Using this method cytokine production and proliferation responses were detected from islet, acinar and spleen extracts. Hence, our method allows the rapid preparation of human tissue lysates in a format that is compatible with the analysis T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.
authors.affiliation: Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 9 Princes St, Fitzroy, Victoria 3065, Australia.
authors.forename: Hayley C
authors.initials: HC
authors.lastname: Moon
authors.forename: Max
authors.initials: M
authors.lastname: Joffe
authors.forename: Helen E
authors.initials: HE
authors.lastname: Thomas
authors.forename: Thomas W H
authors.initials: TW
authors.lastname: Kay
authors.forename: Stuart I
authors.initials: SI
authors.lastname: Mannering
mesh_terms: D049109:Cell Proliferation; D005591:Chemical Fractionation / Q000379:methods*; D016207:Cytokines / Q000096:biosynthesis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007371:Interferon-gamma / Q000737:chemistry / Q000276:immunology; D007515:Islets of Langerhans / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013154:Spleen / Q000737:chemistry* / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D014020:Tissue Extracts / Q000737:chemistry*
chemical_list: D016207:Cytokines; D011506:Proteins; D014020:Tissue Extracts; D007371:Interferon-gamma