Bovine CD8+ T Cell Immunophenotyping

Multi-colour flow cytometry analysis was performed as described previously [19 (link)]. Briefly, cells were incubated with a cocktail of monoclonal antibodies against rabbit CD4 (IgG2a, KEN-4), CD8 (IgG1, 12C.7) and IgM (IgG1, NRBM) on ice for 10 min. Cells were washed and further incubated for 10 min on ice with isotype-specific phycoerythrin (PE)-conjugated rat anti-mouse IgG1 (A85-1, BD) and biotinylated rat anti-mouse IgG2a (R19-15, BD) antibodies. After a third wash, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit T cells (KEN-5), washed with allophycocyanin (APC)-conjugated streptavidin (BD), and suspended in 7-AAD. Antibodies were from AbD-Serotec. Bovine CD8⁺ T cells were identified as previously described [18 (link)], and dead cells detected with BD Horizon Fixable Viability Stain 450 (BD biosciences). Data were acquired using a Fortessa X20 flow cytometer (BD) and analyzed using Flowjo v10.0.7 (Treestar).