Immunophenotyping of Chicken Lymphocytes

For immunophenotyping using flow cytometry, cells were harvested, centrifuged for 10 min at 400× g, washed once with DPBS/EDTA containing 0.05% bovine serum albumin (BSA, Sigma-Aldrich), and stained with different panels of monoclonal antibodies. In this study, CD4+ T cells were stained with mouse anti-chicken CD4-phycoerythrin (PE) or CD4-SpectralRed (SPRD) (CT-4, SouthernBiotech, Birmingham, Alabama, USA). CD8+ T cells were labeled with CD8-allophycocyanin (APC) (CT-8, ThermoFisher Scientific). In addition, human anti-chicken CD25-FITC (AbD13504, Bio-Rad Laboratories, Hercules, California, USA) and CD28-PE (AV7, SouthernBiotech) were used to investigate the activation of CD4+ T-helper cells (CD4+CD25+, CD4+CD28+) and CD8+ cytotoxic T cells (CD8+CD25+, CD8+CD28+). All CD3+ T cells were labeled with the pan-T-cell marker CD3-APC (CT-3, ThermoFisher Scientific). Bu-1+ B cells were stained with Bu-1-FITC (AV20, ThermoFisher Scientific). As a viability marker, 1.5 µL of 1 mg/mL 4′, 6-diamino-2-phenylindole (DAPI, ThermoFisher Scientific) was used. 20,000 vital PBMCs were recorded on a BD FACSCanto™ II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer.