Anti cd49d clone 9f10

Frozen PBMCs were thawed, counted, and resuspended at a density of 15 million live cells per ml in complete RPMI (RPMI with 10% FBS (Gibco) and antibiotics). 100 μl of cell suspension containing 1.5 million cells was added to each well of a 96-well round-bottomed tissue culture plate. The cells were rested overnight at 37 °C in a CO₂ incubator. The next morning, each sample was treated with peptide mega pool (1 μg/ml of each peptide) or single peptide (5 μg/ml) or 0.5% v/v DMSO as negative control in the presence of 1 μg/ml of anti-CD28 (clone CD28.2, BD Biosciences), anti-CD49d (clone 9F10, BD Biosciences), anti-CXCR3 (clone 1C6, BD Biosciences) and anti-CXCR5 (clone RF8B2, BD Biosciences). Peptides were synthesized to 95% purity (Elim Biopharm). All wells contained 0.5% v/v DMSO in total volume of 200 μl per well. The samples were incubated at 37 °C in CO₂ incubators for 2 h, and then 10 μg/ml brefeldin-A was added. The cells were further incubated for 6-8 h.