Although, as described by the authors and in our own analyses, th

Although, as described by the authors and in our own analyses, there are rare populations of CD16+CD8α− NK cells in the peripheral blood of chimpanzees, the data we present here indicate that these populations are often likely to be contaminated by phenotypically FDA approved Drug Library chemical structure and functionally defined CD16+ mDCs. Fresh chimpanzee blood samples were obtained from captive chimpanzees housed at the Yerkes National Primate Research Center, Emory University (supported by NIH grant RR000165). These studies were approved by the

Institutional Animal care and Use Committee of Emory University. The YNPRC is fully accredited by the American Association for Accreditation of Laboratory Animal Care. Cryopreserved samples were analyzed from chimpanzees

originally housed at the Laboratory for Experimental Medicine and Surgery in Primates, New York University, the Coulston Foundation, Alamogordo, New Mexico in biosafety level 2 facilities in accordance with institutional guidelines and Animal Welfare Act guidelines. The protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Chimpanzee PBMCs were isolated from EDTA-treated venous blood by density gradient centrifugation over LSM (MP Biomedicals, Solon, OH, USA) and contaminating red blood cells were lysed using a buy MG-132 hypotonic ammonium chloride solution. After isolation all cells were washed and resuspended in PBS supplemented with 2% FCS (Sigma-Aldrich, St. Louis, MO, USA) for subsequent assays or frozen in a 90% FCS/10% DMSO solution. Cell surface staining was carried out using standard protocols Interleukin-2 receptor for our laboratory as described previously 2 using antibodies listed in Table 1. Intracellular staining for perforin was done using Caltag Fix & Perm (Invitrogen) according to the manufacturer’s recommended protocol. All acquisitions were made on an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). To further confirm the identity

of NK cells and mDCs, we examined their functional responses to NK- and DC-specific ligands ex vivo. PBMCs were resuspended in RPMI 1640 (Sigma-Aldrich) containing 10% FBS and stimulated at an E/T ratio of 2.5:1 with 721.221 cells; PMA (50 ng/mL) and ionomycin (1 μg/mL); poly I:C (100 μg/mL); or medium alone. Anti-CD107a was added directly to each of the tubes at a concentration of 20 μL/mL and Golgiplug (brefeldin A) and Golgistop (monensin) were added at final concentrations of 6 μg/mL, then all samples were cultured for 12 h at 37°C in 5% CO2. After culture, samples were surface-stained using markers to delineate NK cells (CD3, CD8, CD16) and mDCs (HLA-DR, CD11c) as shown in Fig. 1. Cells were then permeabilized using Caltag Fix & Perm and intracellular cytokine staining was performed for IFN-γ, IL-12, and TNF-α. All statistical and graphical analyses were done using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA).

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