Recombinant human chemokines

Cytochalasin D (actin polymerization inhibitor), latrunculin A (actin polymerization inhibitor), jasplakinolide (actin polymerization stabilizer), nocodazole (microtubule polymerization inhibitor), paclitaxel (microtubule polymerization stabilizer), Rac1 inhibitor NSC23766, and pertussis toxin (PTX, Gαi inhibitor) were from Calbiochem (Merck4Biosciences, Burlington, MA, USA), and staurosporine was from Merck Millipore (Burlington, MA, USA). Lipofectamine 2000 and anti-human Rab11 antibody were from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). ¹²⁵I-CCL2 was from PerkinElmer. Recombinant human chemokines, unconjugated anti-human ACKR2 monoclonal antibody, and rat IgG2a isotype control were from R&D Systems. Antibodies for human myosin Vb and Rab4 were from BD Biosciences. Anti-human β-arrestin1, PAK1, and LIMK1 antibodies were from Cell Signaling Technology. Anti-human α-tubulin and vinculin were from Sigma-Aldrich. AlexaFluor 488-conjugated phalloidin, AlexaFluor-conjugated secondary antibodies, and 4′,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes. Horseradish peroxidase (HRP)-conjugated anti-Ig antibodies were from GE Healthcare.

Crude drugs/herbs were dissolved in H₂O at a concentration of 10 mg/ml, and the stock solutions were stored at −80°C. The voucher samples of these extracts were reserved in the Cooperative Research Project of Institute of Natural Medicine at University of Toyama. Ephedrine was purchased from Nichi-Iko (Toyama, Japan) Recombinant human chemokines were purchased from R&D Systems (Minneapolis, MN).

Migration assays were conducted in 5 μm pore Transwell 96-well plates (Corning HTS Transwell 96 well permeable supports). Here, 5 × 10⁵ MAIT cells in 100 μL serum-free complete medium were seeded in the upper chamber of the Transwell. The bottom chamber was filled with 200 μL of recombinant human chemokines at 200 ng/mL (all from R&D systems) in serum-free complete medium. As positive and negative controls, complete medium or serum-free complete medium were used, respectively. After 3 hours at 37°C, cells in the bottom chamber were stained and counted using FACS counting beads (Countbright Absolute Beads for Flow Cytometry, Thermo Fisher Scientific) according to the manufacturer’s instructions. The migration index was the ratio of the number of cells that migrated in the presence of the chemokine to the number of cells that migrated in the presence of the serum-free medium.