Mouse anti human cd3 antibody

Lipo/siRNA complexes (100pmols of siRNA) marked with rhodamine were incubated with PBMC cultured in 48-well culture plates for 4 and a half hours. Paraformaldehyde-fixed cell suspensions were located on microscope slides as described above. Subsequently, samples were stained with primary purified mouse anti-human CD3 antibody (BD Biosciences 1:30, incubated for 1 hour at RT) followed by secondary antibody goat anti-mouse Cy-5 conjugate (Invitrogen 1:100, incubated for 1 hour at RT). WGA conjugated to Oregon Green® 488 (Invitrogen 1:200,) and 1 μg/ml Hoechst 33342 (Invitrogen) were used to counterstain plasma membrane and nucleic acid respectively. In additional stainings the following antibodies were used: FITC (fluorescein conjugated) mouse anti-human CD56 (BD Bioscience 1:30, incubated for 1 hour at RT); APC (allophycocyanin conjugated) mouse anti-human CD86 (BD Bioscience 1:10, incubated for 1 hour at RT); Alexa Fluor 700 mouse anti-human CD19 (BD Bioscience 1:30, incubated for 1 hour at RT); FITC goat anti-mouse secondary antibody (Invitrogen 1:500, incubated for 1 hour at RT); 1 μg/ml Hoechst 33342 (Invitrogen) was used to counterstain nuclei.

Five milliliters of heparinized blood was obtained from 10 OLP patients, respectively. The peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density-gradient centrifugation. Subsequently, CD4+T helper (Th) cells were purified by anti-human CD4 magnetic particles (BD biosciences, USA) on a cell separation magnet (BD Biosciences, USA) according to the manufacturer's instructions. The purified CD4+Th cells were resuspended at a density of 1 × 10⁶ cells/mL RPML 1640 medium (Thermo Scientific HyClone, Beijing, China) supplemented with 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA). For activation of CD4+Th cells, 10 μg/mL of purified mouse anti-human CD3 antibody (BD Pharmingen, USA) and 5 μg/mL of purified mouse anti-human CD28 antibody (BD Pharmingen, USA) were added. To study the effect of IL-23 on the CD4+Th cells, the cells were cultured with or without recombinant human IL-23 (rIL-23, 20 ng/mL; R&D Systems, Minneapolis, MN, USA) for 36 hours. Subsequently, cells and culture supernatant were collected separately for the following intracellular cytokine staining and ELISA detection.