Granulocyte layers were isolated from peripheral blood after density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare, Uppsala, Sweden) and used for eosinophil purifications by negative selection on an AutoMACS Pro separator using the Eosinophil Purification Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Eosinophil purity was >98% for all samples as determined by counting a minimum of 300 cells on cytospin slides stained with Shandon Diff-Kwik stains (ThermoFisher Scientific, Waltham, MA).
Transwell migration assays were performed in 24-well Transwell plates with 5.0 μm pore inserts (Costar, Corning, NY). 500μL of phenol red free RPMI medium (10%FCS), with or without 100 ng/mL recombinant human CCL11 (eotaxin-1, Peprotech, Cranbury, NJ), 100nM 25-HC (Cayman Biochemicals), 100nM 7α,25di-OHC (Cayman Biochemicals) or the GPR183-specific antagonist NIBR189 (50nM) (Tocris) was added to the lower chamber. 100μL of culture medium containing 3 × 105 cells were then added to the upper chamber. After incubation for 3 h at 37°C and 5% CO2, cells were collected separately from the upper or lower chambers and acquired on an LSRII flow cytometer (BD). The proportion of migrating eosinophils was defined as the number of cells in the lower chamber divided by the sum of the cells in the upper and lower chambers and normalized to the positive control of eotaxin-mediated migration.
Transwell migration assays were performed in 24-well Transwell plates with 5.0 μm pore inserts (Costar, Corning, NY). 500μL of phenol red free RPMI medium (10%FCS), with or without 100 ng/mL recombinant human CCL11 (eotaxin-1, Peprotech, Cranbury, NJ), 100nM 25-HC (Cayman Biochemicals), 100nM 7α,25di-OHC (Cayman Biochemicals) or the GPR183-specific antagonist NIBR189 (50nM) (Tocris) was added to the lower chamber. 100μL of culture medium containing 3 × 105 cells were then added to the upper chamber. After incubation for 3 h at 37°C and 5% CO2, cells were collected separately from the upper or lower chambers and acquired on an LSRII flow cytometer (BD). The proportion of migrating eosinophils was defined as the number of cells in the lower chamber divided by the sum of the cells in the upper and lower chambers and normalized to the positive control of eotaxin-mediated migration.