Mouse anti human cd3 clone ucht1

All work was carried out under the Human Tissue Act 2004 and conformed with the principles of the Declaration of Helsinki. Tissue and clinical data were fully anonymized before being handed to researchers. Human left ventricular (LV) tissue was obtained from hearts of end-stage heart failure patients removed during transplant surgery (Research Ethics Committee approval: 09/H0504/104+5; Biobank approval number: NP001-06-2015). Donor hearts unsuitable for transplantation were used as controls (Research Ethics Committee approval: 16/LO/1568). Informed consent was obtained from patients or next of kin. For detection of CD3⁺ and CD8⁺ T cells in heart failure hearts, frozen sections of human heart tissue were stained with mouse anti-human CD3 (clone UCHT1 at 1:100), and mouse anti-human CD8 (clone HIT8a at 1:100) with Alexa Fluor 488 goat anti-mouse IgG (poly4053 at 1:500; all purchased from BioLegend, London, UK).

For staining, cells were first detached with trypsin (Life Technologie) and washed twice in PBS containing 2% FCS (Biowest) and 0.1% sodium azide (NaN3, Sigma-Aldrich). Next, antigen nonspecific binding was prevented by prior incubation of cells with 10% mouse serum (Fitzgerald). Cells were next incubated with combinations of pacific blue, fluorescein isothiocyanate (FITC), and allophycocyanin (APC) –conjugated mouse anti–human antibodies. The following monoclonal antibodies were used: mouse-anti-human CD3 (Clone UCHT1, Biolegend), mouse-anti-human CD19 (Clone HIB19, BD Biosciences), mouse-anti-human CD56 (Clone B159, BD Biosciences), and mouse-anti-human HLA-ABC (Clone W6/32, Biolegend). Cells were acquired on FACSCanto II and analyzed using FACS Diva Version 6.13 (BD Bioscience) or FlowJo version 7.6.5 software. Data were analyzed using GraphPad Prism 5.