Cd3apc cy 7 clone 17a2

Blood, spleen, peyer’s patch, bone marrow and mesenteric lymph nodes were collected and processed to single cell suspensions. Cells were prepared for surface staining with the indicated antibodies. To identify the varying B cell responses fluorochrome-conjugated antibodies against B220 PerCP (clone RA3-6B2), CD19 PE/Cyanine7 (clone 6D5), CD86 PE (clone PO3), CD80 APC (clone 16-10A1), GL7 FITC (clone GL7) and CD95 (Fas) PE/Cyanine7 (clone SA367H8) (Biolegend^®) were used, and CD4 FITC (clone GK1.5), CD3APC-Cy^™7 (clone 17A2), PECy7 CXCR5 BV421 (clone 2G8) (BDBioscience^™) and PD1 PE-Cyanine7 (BDBioscience^™) were used to assess T cell responses. Cells were stained for 30 minutes and then washed before analysis using a BD FACS Canto II instrument (BD Bioscience, San Diego CA). Data analyses were performed using the FlowJo software (BD Bioscience).

Cells were collected after 72 h of incubation from either the lymphocyte proliferation assay or from the co-culture assay. Non-adherent cells were transferred to a new plate and 5 μg/mL of Brefeldin A was added. Four h later, plates were centrifuged for 3 min at 500 × g and supernatants were removed. After blocking of the Fc γ receptors with CD16/CD32 mouse BD Fc block (clone 2.4G2, BD Biosciences), cells were labeled with cell surface marker monoclonal antibodies (mAb) and conjugated intracellular cytokine mAb as recommended by BD Biosciences. The following mAb conjugates were used: CD3-APCCy7 (clone 17A2, BD Biosciences), CD4-AF700 (clone RM4-5, BD Biosciences), CD8-PerCPCy5.5 (clone 53-6.7, BD Biosciences), IFNγ-PE-CF594 (clone XMG1.2, BD Biosciences), TNF-α-Brilliant violet 421 (clone MP6-XT22, BioLegend), IL17A-PE/Cy7 (clone TC11-18H10.1, BD Biosciences). Aqua Viability Dye (Molecular Probes/Invitrogen) was added to distinguish live and dead cells. Cells were acquired using an LSRII flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). Results were analyzed using FlowJo software (Tree Star).