Pecy7 conjugated anti cd56

Procedures were previously reported [5 (link)]. Isolated PBMCs were washed and then incubated overnight (37°C, 5% CO₂) in RPMI 1640 supplemented with 10% fetal bovine serum. Cells were washed and treated with the respective concentrations of 1615EpCAM133, EpCAM16, anti-EpCAM scFv, IL-15 (NCI derived), anti-CD133 scFv and incubated for 10 minutes at 37°C with 5% CO₂. FITC-conjugated anti-human CD107a monoclonal antibody (mAb) (LAMP-1) (BD biosciences, New Jersey, CA, USA), was added and further incubated for 1 hour. GolgiStop (1:1500) (BD Biosciences, San Jose, CA, USA) and GolgiPlug (1:1000) (BD Biosciences, San Jose, CA, USA) were added and cells were further incubated for 3 hours. Cells were washed and stained with the following mAb from BioLegend, San Diego, CA, USA: PE/Cy7-conjugated anti-CD56; APC/Cy 7-conjugated anti-CD16; PE-CF594-conjugated anti-CD3. After incubation for 30 minutes, cells were fixed with 2% paraformaldehyde. For intracellular staining, Pacific Blue-conjugated anti-human IFN-γ (BioLegend, San Diego, CA, USA) was used after permeabilization with permeabilization buffer (BD Biosciences, San Jose, CA, USA). After incubation for 15 minutes cells were washed and evaluated by FACS analysis using a LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).

Whole blood samples were collected from each subject by venipuncture, and density gradient centrifugation was used to extract PBMCs. CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), monocytes (CD3−CD14+), natural killer (NK) cells (CD3−CD56+), and B cells (CD3−CD19+) of HCs were sorted from PBMCs using a BD FACS Aria flow cytometer. The following monoclonal antibodies (mAbs, Biolegend) were used: PerCP-conjugated anti-CD3, FITC-conjugated anti-CD4, PE-conjugated anti-CD8, PE-CY7-conjugated anti-CD14, PE-CY7-conjugated anti-CD56 and PerCP-conjugated anti-CD19. STEMCELL was used to sort CD8+ T cells from HIV-infected patients, and the sorting purity was detected by flow cytometry. CD8+ T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (HyClone), 1% penicillin–streptomycin (Gibco) and IL-2 [30 (link), 31 (link)] (30 U/ml, Sigma). Transfection of siRNA and controls (Invitrogen) was performed with Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen). Briefly, cells were transfected with 100 pmol of eIF3d siRNA or 75 pmol of eIF3d siRNA plus 75 pmol of SOCS-7 siRNA. Transfection efficiency was measured by real-time PCR after 48 h of transfection.