Pe cy7 conjugated anti cd14

Freshly isolated monocytes were stained with PE/Cy7‐conjugated anti‐CD14 (301814; Biolegend).³⁴ For ROS measurement, monocytes were seeded at 0.5 × 10⁶ cells/mL and treated with H₂O₂, irradiation, nutlin‐3, or DMSO. After the indicated time periods, cells were washed with PBS and resuspended with 10 µM H2DCFDA (D399; Invitrogen) in the dark for 30 min at 37°C. Cells were then washed for twice. Fluorescence was measured and data of mean fluorescence intensity were analyzed by Kaluza (Beckman Coulter, 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.