Histopaque cushion

Blood was collected by femoral venipuncture into EDTA, heparin, and clot-activating vacutainer tubes (BD Biosciences). The EDTA plasma and serum tubes were centrifuged at approximately 1,300g at 4°C for 10 minutes; afterward, the upper layer was collected. For isolation of PBMCs, heparin-treated blood and the spun EDTA pellet were diluted with PBS and carefully layered onto a Histopaque cushion within Accuspin tubes (Sigma-Aldrich). The tubes were centrifuged at approximately 800g room temperature for 15 minutes, and the resulting buffy coat was collected. Cells were washed once in R10 (RPMI medium [Gibco] supplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin solution, and 1% l-glutamine) and treated briefly with ACK lysing buffer (Gibco) to remove any contaminating erythrocytes. PBMCs were then centrifuged at approximately 250g for 10 minutes to eliminate residual thrombocytes, washed twice with R10 medium, and enumerated with a TC20 Automated Cell Counter (Bio-Rad). Cells were cryopreserved in 10% DMSO in FBS. Before flow cytometry, cryopreserved PBMCs were thawed rapidly in a 37°C water bath (BD Biosciences).