Anti cd3 alexa flour 700

Anti-CD16/CD32 (BD, 2.4G2 [anti-FcγR]) was used as a blocking reagent and PI-PECF594 (Invitrogen, CAT P3566) was used to identify dead cells. The following antibodies and reagents were used for germinal center B-cell staining: anti-IgD-BV-605 (BioLegend,11-26c.2a), anti-B220-APCeFlour780 (eBioscience, RA3-6B2), anti-CD38-Alexa 700 (eBioscience, 90), and anti-CD95-PE-CY7 (BD, Jo2). For T follicular helper cell (Tfh) staining, anti-CXCR5 (BD, 2G8), anti-rat IgG (fab’) Alexa-647 (Jackson Immuno Research, Code:712-606-153). 2W-specific T-cells were identified using a 2W:I-Ab tetramer labeled with PE (50959 I-A(b)) or BV421(50960 I-A(b) (NIH Tetramer Core Facility), and the following antibodies: anti-CD3-Alexa flour 700 (BioLegend, 17A2), anti-CD4-FITC (BioLegend, RM4-5), anti-B220-APCeFlour780 (BioLegend, RA3-6B2), and anti-PD1-PE-CY7 (eBioscience, J43). The Click-iT™ Plus EdU Alexa fluor 647 Flow cytometry kit was used for EdU staining of newly synthesized DNA (Invitrogen, CAT C10634). Porcine cardiac myosin (Sigma-Aldrich, CAT: M0531) and a cardiac myosin monoclonal antibody (ThermoFisher, CAT: MA1-26180) was used for investigation of myosin-reactive antibody responses.

Cell surface staining was processed using whole blood lysis method. Freshly drawn heparinized peripheral blood samples were labeled with monoclonal antibody (mAb) panel for T lymphocyte subsets [anti-CD3-AlexaFlour700, -CD4-APC, -CD8-APC/CY7, -CD45-BV510, -CD45RA-FITC, -CD45RO-PE, -CCR7-PE and -HLA-DR-BV711 (all from Biolegend, San Jose, CA, USA)], mAb panel for B lymphocyte subsets [anti-CD19-APC, -CD24-PE, -CD27-PE/CY7, -CD38-AlexaFlour700, -CD45-BV510, -CD138-BV421 and -IgD-FITC (all from BD-Biosciences, San Jose, CA, USA)], and with mAb panel for Treg [anti-CD3-AlexaFlour700, -CD4-APC, -CD25-PE, -CD45-BV510, -CD127-BV421 (all from BD-Biosciences, San Jose, CA, USA)]. Following incubation, erythrocytes were lysed using FACS Lysing Solution (BD Biosciences, San Jose, CA, USA), and at least 10.000 cells were collected in CD45⁺ lymphocyte gate. The data were acquired on a NovoCyte flow cytometer (Agilent Technologies, USA) and analyzed using the NovoExpress operating system software (Agilent Technologies, USA).