Expression of arginase I and inducible nitric oxide synthase in the peripheral blood and lymph nodes of HIV-positive patients

ZHANG, NAICHUN; DENG, JIANNING; WU, FENGYAO; LU, XIANGCHAN; HUANG, LEI; ZHAO, MIN

Detection of the expression levels of Arg I and iNOS using flow cytometry. The mononuclear cells from the peripheral blood or LNs were added to three tubes (2-5x10 5 cells/tube), and were incubated with anti-CD3-fluorescein isothiocyanate (cat. no. 349201; monoclonal mouse IgG1; clone SK7; BD Biosciences) and anti-CD8-peridinin chlorophyll protein (cat. no. 347314; monoclonal mouse IgG1; clone SK1; BD Biosciences) and permeabilized using a Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences). Subsequently, the three tubes (Arg, iNOS and isotype control) were divided into three groups and either anti-hArginase1-phycoerythrin (cat. no. IC8026P; monoclonal mouse IgG2B; clone 658922; R&D Systems), anti-iNOS antibody (cat. no. ab15323; rabbit polyclonal; Abcam) or isotype control antibody (cat. no. IC0041P; monoclonal mouse IgG2B; clone 133303; R&D Systems) was added to one tube containing peripheral blood mononuclear cells and one tube containing LN mononuclear cells. 1XBD Perm/Washâ„¢ Buffer from the kit was used for washing. The cells in the iNOS group were incubated with goat anti-rabbit IgG-allophycocyanin (cat. no. ab130805; polyclonal; Abcam) for 15 min at 20ËšC and fixed in 1% paraformaldehyde for 24 h. Flow cytometry was performed within 24 h using a FACSCanto II flow cytometer (BD Biosciences). FlowJo software (version 7.6.1; FlowJo LLC, Ashland, OR, Canada) was used to analyze the data. The lymphocytes were gated based on forward scatter/side scatter, which were further separated into CD8 + T cells (CD3 + /CD8 + ) and CD4 + T cells (CD3 + /CD8 -), and the expression levels of Arg I and iNOS were analyzed.