T cell migration assays

T cell migration assays 

Human peripheral blood-derived mononuclear cells were isolated from author by centrifugation on a Ficoll–Hypaque gradient, washed with Hank’s balanced salt solution and suspended in RPMI-1640 complete media (with100 μg /ml streptomycin). Peripheral blood-derived mononuclear cells were activated for 2 days with 5 μg/ml anti-human CD3 Ab (eBiosciences) and 2 μg/ml anti-CD28 Ab (eBiosciences), then expanded for 5 days to generate activated T cells with recombinant human IL-2 (Novartis). In vitro migration assays were performed in a Transwell system (Corning) with a polycarbonate membrane of 6.5-mm diameter with a 3μm pore size. These activated T cells were loaded into the top chamber of transwell inserts. In the bottom well, RPMI-1640 medium containing culture supernatant from MDA-MB-231 cells with ENMD-2076 treatment (5μM) for 48 hour was added. Plates were incubated at 37 ℃ for 2-6 h; the contents of the lower chamber were collected; and the percentage of CD8+ T cells present in the bottom chamber was determined by FACS (LSR II, BD Biosciences).

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