Transwell culture inserts

We used the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega) for the assessment of cell proliferation. Migration and invasion assays were performed by the Boyden chamber method. For the invasion assay, 8-μm-pore filters set in Transwell culture inserts (BD Biosciences) were coated with Matrigel. Cells (3 × 10⁴ cells/insert) were placed in the upper chamber in a low-serum medium, D/F (0.5% FBS), and the lower chamber was filled with a high-serum medium, D/F (10% FBS). Twelve hr later, the cells that had passed through the membrane were stained with hematoxylin and eosin (H&E) (Muto Pure Chemicals, Tokyo). Each Transwell insert was microscopically imaged in five distinct regions at 100 × magnification in triplicate. The numbers of cells that had migrated to the five non-overlapping regions were counted using BZ-analysis software (Keyence, Osaka, Japan) and summed as the total cell number and are presented as the average of three independent experiments.

The 24-well Transwell culture inserts (8 μm, BD Biosciences, USA) were used for cell migration assay according to the manufacturer’s instructions. THP-1-derived macrophages (1 × 10⁵) were resuspended in 100 μl of serum-free RPMI-1640 medium and seeded into the upper compartment of each well. RPMI-1640 medium (600 μl) with 10% FBS and 50% CAL-27 CM was added into the lower chamber of the plate. RPMI-1640 medium with 10% FBS but without CAL-27 CM was used as control. IL-6 NAb/IsoAb (10 μg/ml, R&D, USA) or HMGB1 NAb/IsoAb (1 μg/ml) was added into CAL-27 CM as indicated. After incubation at 37°C in 5% CO2 for 24 h, the migrated cells were fixed using 10% formalin and stained with eosin. Cell numbers of five random fields were counted, and images were taken.

In vitro cell migration was examined using Transwell culture inserts (BD Biosciences, Franklin Lakes, NJ, USA) with 8 μm pore filter inserts for 24-well plates. Briefly, 5 × 10⁵ cells suspended in serum-free DMEM were added to the inserts, the upper chamber, which was placed in a 24-well culture plate. FBS was added to the lower chamber at the final concentration of 2% as a chemoattractant. After 6 h, the upper insert was removed, washed and the non-filtered cells were gently removed with a cotton swab. Filtered cells located on the lower side of the chamber were stained with crystal violet, photographed and counted using Image J ver.1.48 (http://imagej.nih.gov/ij/).