Fig 2: Gating strategy of bovine monocyte subsets based on relative CD14 and CD16 expression.(A) Three-color immunofluorescence of bovine MNC with mAbs to CD172a, CD14 and CD16 defines three monocyte subsets in peripheral blood. Viable (propidium-iodide-negative) mononuclear cells, based on forward and side scatter characteristics, were gated on CD172a-positive cells. Dot plots of CD14 and CD16 expression display classical monocytes (CD14+CD16-, upper left), intermediate monocytes (CD14+CD16+, upper right) and nonclassical monocytes (CD14-CD16+, lower right). (B) Monocytes were gated in a side scatter/CD172a dot plot, identifying monocytes as CD172a-positive cells. Histograms show that CD172a-positive cells do not express common markers of B cell (CD21), T cell (CD2, CD4 or CD8), NK cell (NKp46) or blood ?d T cell (WC1). Data are shown for cells from one of five tested animals.

Fig 3: Purification of bovine monocyte subsets.(A) Bovine cM (CD14+ CD16-), intM (CD14+ CD16+) and ncM (CD14- CD16+) subsets were separated from bovine MNC using a two-step MACS procedure as described in material and methods. Separated subsets were labeled with monoclonal antibodies to CD14 and CD16 to identify their purity. Representative examples from 5 independent experiments are shown. (B) Microscopic images of separated monocyte subsets are shown with magnification 100 after staining with acridine orange. (C) Bovine MNC were labeled with monoclonal antibodies to CD14 and CD16 to identify three monocyte subsets. Gated monocyte subsets were presented in FSC against SSC. The median of FSC and SSC of the individual subsets was measured (n = 6 cows) and presented as mean ± SEM. Statistical analysis of significance were performed using ANOVA and Bonferroni’s correction for normally distributed data. *(P<0,01).

Fig 4: Changes in the monocyte subsets of PBMCs in Holstein and Jersey steers subjected to heat stress. Flow cytometry analysis to identify monocyte subset population. A CD172a+ monocyte cells were sorted from total PBMCs and B a dot plot depicting the three monocyte subsets in monocytes (CD172a+): classical CD14+CD16-, intermediate CD14+CD16+, and non-classical CD14-CD16+ monocytes. MT indicates moderate THI condition and HT indicates high THI condition