title: A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.
abstract: In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: La Muraglia
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: O'Neil
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Madariaga
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: S G
authors.initials: SG
authors.lastname: Michel
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Mordecai
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA; Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Allan
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA; Division of Cardiac Surgery, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: J C
authors.initials: JC
authors.lastname: Madsen
authors.affiliation: Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
authors.forename: I M
authors.initials: IM
authors.lastname: Hanekamp
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, MA, USA. Electronic address: preffer@helix.mgh.harvard.edu.
authors.forename: F I
authors.initials: FI
authors.lastname: Preffer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D013234:Stem Cells / Q000276:immunology*; D013552:Swine; D013556:Swine, Miniature; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
keywords: Cell-mediated lympholysis; Chromium-51; Cytotoxic lymphocyte; Imaging flow cytometry; Major histocompatibility complex; Transplantation