PBMC immunophenotyping by flow cytometry

The following antibodies were used to label the different PBMC subpopulations: anti-ovine CD4 (clone 44.38), CD8 (clone 38.65), and WC1 (clone 19.19), anti-human CD14 (clone TÜK4) and CD16 (clone KD1) (all from Bio-Rad), and anti-bovine B cell marker (clone BAQ44A) (Kingfisher Biotech). PBMC flow cytometry was performed as described in (1 (link)). Briefly, PBMCs were washed twice in staining buffer (PBS + 2% FBS + 0.02% sodium azide) and stained for 20 min on ice with the appropriate fluorochrome-conjugated surface antibodies. In the case of the unconjugated anti-bovine B-cell marker, a rat anti-mouse IgM FITC (clone II/41, BD Biosciences) was used as secondary antibody. Samples were run on a FACSCalibur (Becton Dickinson) flow cytometer. Dead cells were excluded from the analysis by 7-AAD staining (BD Biosciences) and gating was performed using the appropriate isotype controls. Analysis was performed using FlowJo software (TreeStar Inc.).