Hts transwell 96 well permeable support system

Migration assays were carried out using the Corning HTS Transwell 96-well permeable support system with 5.0 µm pore size polycarbonate membrane. The bottom wells were filled with 200 µL migration buffer alone (RPMI-1640 medium with 0.1% fatty acid-free BSA and 10 mM HEPES) or migration buffer containing either 10 nM S1P (Sigma-Aldrich) or 20% B16 tumor-conditioned media (TCM). To prepare B16 TCM, single-cell suspensions from B16 tumors grown in WT mice were plated at 1 × 10⁶/mL in serum-free RPMI-1640 media for 24 hours (Deng et al., 2012 (link)). Media was collected, 0.22 µm filtered, aliquoted and stored at −80°C. To deplete lipids, TCM was incubated with dextran-coated charcoal (T-70, Sigma) at 10 mg/mL for 30 min. Total splenocytes harvested from tumor-bearing mice were stained with APC-CD3 and PE-CD4 antibodies. Cells were then washed three times and resuspended in migration buffer to a final concentration of 1 × 10⁷/mL. In some experiments, cells were then incubated with or without 5 µM AZD1480 (AstraZeneca) at 37°C for 30 min. 50 µL of cells was added into each top well and allowed to migrate at 37°C for 1–2 hrs. Migrated cells were fixed, permeabilized, and stained with FITC-Foxp3 antibody, and migrated cells in the bottom wells were counted by flow cytometry (Accuri, BD Biosciences). Triplicates were performed for each condition.