Mouse anti human cd4 pe

Cell sorting was performed to separate CD3+CD4+ and CD3+CD8+ lymphocyte populations. The absolute numbers of the PBMCs were determined using DXH 500 (Beckman Coulter, CA, USA) equipment in order to adjust the volume of each antibody. FITC mouse anti-human CD3, PE mouse anti-human CD4, and PerCP-Cy 5.5 mouse anti-human CD8 antibodies (BD Pharmingen, CA, USA) were used according to the manufacturer’s instructions and incubated for 15 min at room temperature, in the dark. Cells were further washed in PBS, centrifuged at 450× g for 5 min, and pelleted cells resuspended in PBS until sorting using a FACSAria III^TM from Becton Dickinson, CA, USA. About 10,000 events were acquired to define the gating strategy using morphologic and fluorescence parameters. Using a dot-plot from FSC (forward light scatter) vs. SSC (Side scatter), lymphocytes were gated and, after excluding doublet, lymphocytes subsets were identified by gating CD3+CD4+ and CD3+CD8+, and non-CD3+. Monocytes were initially selected by morphologic parameters, doublets were excluded, and monocytes were identified by CD4 positivity since we did not use a specific monocyte marker. No major differences were observed in the percentage of lymphocyte subpopulations and monocytes obtained from the different groups (Figure S1).

At days 2, 4, 8, the lymphocytes suspension were collected by concentrated at 1000 revolutions/min for 10 min, then the precipitate was resuspended with 1 ml 0.9% physiological saline, after centrifuged at 1000 revolutions/min for 10 min, the precipitate was resuspended with 150 μl 0.9% physiological saline, and divided into three groups. One group as isotype control was added FITC mouse IgG2α (5 μl, BD), PE mouse IgG1 (5 μl, BD) and PerCP-CyTM 5.5 mouse IgG1 (1 μl, BD), the experiment group was divided into two group and, respectively, added FITC mouse anti-human CD3 (5 μl, BD), PE mouse anti-human CD4 (5 μl, BD), PerCP-CyTM5.5 mouse anti-human CD8 (1 μl, BD) and FITC mouse anti-human CD19 (5 μl, BD), PE mouse anti-human CD138 (5 μl, BD). The three groups were all incubated at room temperature for 15 min, then resuspended with 1 ml 0.9% physiological saline and centrifuged at 1000 revolutions/min for 10 min. Finally, the precipitate was resuspended with 0.2 ml 0.9% physiological saline and prepared to analysis using BD FACSCanto II. The experiment was repeated for 3 times.

HEK293FT, FaDu, and Hela cell lines were purchased from the American Type Culture Collection. The cells were cultured with Dulbecco’s modified Eagle medium with 10% fetal bovine serum and 100 μg/mL penicillin–streptomycin. Polyethylenimine (PEI) used as a cell transfection reagent was purchased from Sigma (St. Louis, MO, USA). Antibodies used in this study included Cy3-conjugated goat anti-mouse IgG (ProteinTech, SA0009-1), PE mouse anti-human CD4 (BD Pharmingen, 55347), and FITC mouse anti-human CD8 (BD Pharmingen, 555366).