Figure 1

Gating strategies and myeloid population identification. The expression of CD16 and CD14 was determined by single and two colour flow cytometry in freshly isolated bovine PBMC. The cells were gated to eliminate dead cells (A) and doublets (B). Threshold levels which determined positivity for CD14 and CD16 were set with no antibody control (C, D and E) and FMO controls (F, G) were used to determine quadrant position and fluorescence intensity for subsequent analysis. Cells above fluorescence of 400 for FITC (CD16) and above 300 for AF647 (CD14) were determined as positive for the respective molecules. In order to further define the CD16⁺ populations triple staining with CD14, CD16 and NKp46 (I) or CD14, CD16 and CD172a (J) was carried out. Within PBMC gated as CD14⁻CD16^+/++ (rectangular gate; H) the majority of NKp46⁺ NK cells expressed CD16 at a fluorescence intensity of ~800-10 000 (I). The majority of cells which were CD172a negative (J) expressed CD16 at fluorescence intensity ~800-10 000 corresponding with the NKp46⁺ population. A subpopulation of CD14⁻ cells which were NKp46⁻ and CD172a⁺ were observed at fluorescence intensities ≥10 000 and were gated in further studies as a separate population for analysis. Data shown are for one representative animal out of six.