Anti cd8 fluorescein isothiocyanate fitc

Cyclophosphamide monohydrate and levamisole hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), concanavalin A (Con A), lipopolysaccharide (LPS), and Roswell Park Memorial Institute (RPMI)-1640 medium were obtained from Sigma-Aldrich (St. Louis, MO, United States). Cytokines and IgG enzyme-linked immunosorbent assay (ELISA) kits were purchased from ELK Biotechnology (Wuhan, China). Red blood cell (RBC) lysing buffer, fetal bovine serum (FBS), and penicillin-streptomycin (P/S) were purchased from Gibco Life Technologies (New York, NY, United States). Dimethyl sulfoxide (DMSO) was obtained from Duksan Pure Chemical Co., Ltd. (Kyungkido, South Korea). Phosphate-buffered saline (PBS) was provided by Welgene, Inc. (Gyeongsangbuk, South Korea). Anti-CD4⁺ [phycoerythrin (PE)] and anti-CD8⁺ [fluorescein isothiocyanate (FITC)] antibodies were supplied by BD Biosciences (San Diego, CA, United States).

Peripheral blood mononuclear cells (PBMCs) were separated from whole blood samples using Ficoll-Hypaque centrifugation (Amersham Pharmacia, Uppsala, Sweden). Isolated PBMCs were stained using anti-CD8-fluorescein isothiocyanate (FITC; BD Pharmingen, CA) and CD223-phycoerythrin (PE) mouse anti-human fluorescent monoclonal antibody (R&D Systems, Inc., Minneapolis, MN) for 30 minutes at room temperature. The percentage of CD223 (LAG-3) expression in gated CD8⁺ lymphocytes was detected using a Becton Dickinson FACS system and CELLQuest software (BD Bioscience).

Two weeks after the last immunization, the mice were euthanized, and the spleens were removed. The cell suspension was obtained by macerating the spleen and treating the cells with ammonium chloride and potassium (ACK buffer) to lyse the erythrocytes. The spleen cells were stimulated for 16 h, at 37°C in a 5% CO₂ atmosphere and in the presence of brefeldin-A, with a synthetic peptide (GenScript) corresponding to the CD4 (AMQMLKETINEEAAE) or CD8 (AMQMLKETI) T cell-specific epitope of the HIV gag p24 protein, at a final concentration of 5 µg/mL or 10 µg/mL, respectively. Following incubation, cells were washed twice in PBS supplemented with 2% of fetal bovine serum (FBS) and surface stained with anti-CD8 fluorescein isothiocyanate (FITC) and anti-CD4 Cy (BD BioSciences). Next, the cells were washed, fixed with paraformaldehyde and permeabilized with saponin, and then intracellularly stained for IL-4 or IFN-γ phycoerythrin (PE) (BD BioSciences). FACS analysis was performed on a BD FACSCalibur flow cytometer, and the results were analyzed using FlowJo (Tree Star) software.