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Figure 2 | Veterinary Research

Figure 2

From: An in vitro model to assess the immunosuppressive effect of tick saliva on the mobilization of inflammatory monocyte-derived cells

Figure 2

Adaptation of the reverse-transmigration in vitro model with bovine cells to recapitulate monocyte-derived cells mobilization from blood to the draining lymph node under inflammatory condition. A-D Schematic representation of the in vitro differentiation of blood monocytes into macrophages and Mo-DCs using the reverse-transmigration model. E-I Two-color flow cytometry assay of monocyte-derived cells collected from monocyte/BAEC cultures grown on collagen-coated transwells according to the reverse-transmigration protocol. Non-adherent and low-adherent cells within the top and bottom sections of the transwell were processed for cytometry analysis. Left contour plots show the conditions in the absence of any exogenous stimulus whereas right contour plots show conditions with zymosan. Cells were labeled with anti-SIRP1-α to discriminate monocyte-derived cells from endothelial cells. The expression of MHCII within the SIRP1-α+ population was also determined to discriminate within monocyte-derived cells potential macrophages (MHCII+) and potential Mo-DC (MHCIIhi) (E). The percentages of SIRP1-α+MHCII+, SIRP1-α+MHCIIhi and BAECs in each representative dot plot are indicated (E). F Comparison showing the relative number of SIRP1-α+MHCII+ cells in the bottom of the transwell in conditions with or without zymosan stimulation. G Comparison showing the relative number of SIRP1-α+MHCII+ cells in the top section of the transwell in conditions with or without zymosan stimulation. H Comparison showing the relative number of SIRP1-α+MHCIIhi cells in the top section of the transwell in conditions with or without zymosan stimulation. i Comparison showing the relative expression level of MHCII in SIRP1-α+MHCII+ cells in the top section of the transwell in conditions with or without zymosan stimulation. F-I The value of the control condition was arbitrarily set to 1. Data are presented as the mean ± SD based on four independent experiments each including at least three transwells per condition. Statistically significant differences between the two groups were determined by the Student’s t test, *P < 0.05 and **P < 0.005.

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