Chemotaxis assay

HTS Transwell-96 permeable support with 3.0-μm pore polycarbonate membrane (3386; Corning) were used to assess migration of CCR6⁺ or CXCR3⁺ cells isolated from human or macaque PBMCs or of murine CD4⁺ splenocytes isolated from C57BL/6 mice. Briefly, after an overnight culture, 15 × 10⁴ cells were diluted in chemotaxis buffer (RPMI 1640 supplemented with 0.05% pasteurized human albumin) and added to the upper wells. For assessing chemotaxis of human or macaque cells, recombinant human CCL20 or CXCL10 (R&D Systems) was diluted in chemotaxis buffer and added to the bottom wells. For assessing chemotaxis of murine cells, recombinant mouse CCL20 or CXCL12 (R&D Systems) was diluted in chemotaxis buffer and added to the bottom wells. For cofilin inhibition assays, 250 nM OA was added to the chemokine in the bottom wells. Migration was allowed to proceed for 90 min at 37°C in 5% CO₂. Migrated cells were collected from the bottom well, stained with CD3-allophycocyanin-CY7, CD4-PercP-Cy5.5, and CD20-FITC, suspended in a fixed volume, and counted at constant speed with a BD LSRFortessa. Chemotaxis index was calculated as the fold increase in the number of migrated cells in response to chemokine over the spontaneous cell migration in response to chemotaxis buffer alone.