Migration was measured in 48-well micro Boyden chambers equipped with a 5-μm–pore size cellulose nitrate filter, which separated the upper and the lower chambers (Neuroprobe, Gaithersburg, MD). BMDMs were resuspended in RPMI 1640, 0.5% bovine serum albumin (BSA) (1 × 106 cells/ml). After a migration period of 4 hours, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin. The migration depth of the cells into the filters was quantified by microscopy, measuring the distance (in micrometers) from the surface of the filter to the leading front of three cells. Data are expressed as a chemotaxis index, which is the ratio between the distance of directed migration and random migration of monocytes into the nitrocellulose filters.

Alternatively, THP1 cell migration was assessed using the Neuroprobe ChemoTx System (Neuroprobe, Gaithersburg, MD). THP1 cells were loaded with calcein-AM and then migrated toward pepducins or chemokines in the lower wells of the chamber. In inhibition experiments, THP1 cells were preincubated with the pepducins and then migrated toward chemokines or GPR35 agonists.

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