Anti human il 8

A total of 6 × 10⁴ tumor cells were plated into the bottom well of 24-well Transwell® plates (Corning Inc., Corning, NY) and incubated with antibodies (10 μg/mL). Twenty-four hours later, 2 × 10⁵ PBMCs from healthy donors were added to the top well (5 μm) of the Transwell plate and incubated for additional 4 hrs. Wells containing no tumor cells in the bottom chamber were used to quantify spontaneous migration. In some experiments, anti-human MCP-1 or anti-human IL-8 (R&D Systems, Minneapolis, MN) was added 30 min before the addition of PBMC. The total number of immune cells that had migrated into the bottom well was quantified by FACS using polystyrene beads (Polysciences, Warrington PA). Specific migration was calculated by 100 × ((number of cells that migrated toward tumor cells − number of cells that migrated spontaneously)/number of PBMCs seeded). In some experiments, the supernatants from antibody-treated tumor cells were measured for cytokine production by Luminex, and immune cells that had migrated were phenotyped by collecting migrated cells and staining them with antibodies from BD Biosciences: CD3-FITC, CD56/CD16-PE, CD4-PerCP Cy5.5, CD14-PE Cy7, CD11c-APC, and CD20-APC Cy7. Analysis was performed on a FACSCanto flow cytometer.