Ccl28

Chemotaxis assays with purified splenocytes and MLN lymphocytes (MLNLs) were performed as previously described (28 (link)). A microchamber Transwell system with 3-μm pores (Corning Costar, USA) was used in the chemotaxis assays. In brief, 2 × 10⁶ cells/well (in 300 μl complete RPMI 1640 medium) were plated in triplicate in the upper chambers, and 600 μl complete RPMI 1640 medium with or without (negative control) 30 ng/ml CCL19 or 500 ng/ml CCL28 (R&D Systems, USA) was added into the lower chambers. The plates were incubated at 37°C for 90 min, and the number of migrated cells was counted using a Bio-Rad automated cell counter. The fold change in migration was calculated as migrated cell number in testing wells divided by migrated cell number in negative-control wells.

Magnetically enriched SSEA4⁺ cells were plated overnight (3 × 10⁵ cells/well) onto a 6-well plate and stimulated with CCL28 (200 ng/mL, R&D Systems, Bio-Techne) and/or hydrogen peroxide (5 μM, MilliporeSigma) for 7 days. Fresh conditioned medium was replaced once during this period. On day 7 cells were counted and processed for flow cytometric analysis or quantitative PCR.