The assay was performed and analyzed as described [30] (link). Briefly, cells from the afferent lymph of sheep or mouse mononuclear splenocytes were incubated in RPMI containing 0.5% bovine serum albumin (Invitrogen) for 1 h. 5–10×105 cells in 100 µl were added to 5-µm-pore-sized, 24-well tissue culture inserts (Corning costar). Recombinant mouse CCL21 (R&D Systems) and recombinant human or mouse CXCL12 (R&D Systems) were titrated in triplicates, and cells were allowed to migrate for 1.5 h. Lymphocytes in the migrated and input wells were quantified using a bead standard (15 µm polystyrene beads, Polyscience Inc.) combined with the flow cytometric analysis of T cell subsets.
To test the effect of AMD3100 on T cell chemotaxis, mouse splenocytes were incubated with 5 or 25 µM AMD3100 (Sigma-Aldrich) for 15–30 min in RPMI1640 with 0.5% BSA, before subjecting them to a chemotaxis assay in the presence of AMD3100.
To test the effect of AMD3100 on T cell chemotaxis, mouse splenocytes were incubated with 5 or 25 µM AMD3100 (Sigma-Aldrich) for 15–30 min in RPMI1640 with 0.5% BSA, before subjecting them to a chemotaxis assay in the presence of AMD3100.
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