Apc labeled anti cd3

PBMCs and PFMCs were isolated from heparinized venous blood and pleural fluid by Ficoll density-gradient centrifugation and cultivated in 96-well plates (10⁶ cells per well) with RPMI-1640 (Invitrogen, Carlsbad, CA, USA) medium and completed with 0.3 mg/mL L-glutamine, 5 mM HEPES buffer, 100 μg/mL gentamycin, and 10% inactivated fetal bovine serum (FBS). To evaluate PD-1, PD-L1, and PD-L2 expressions on T cells and monocytes/macrophages, freshly isolated PBMCs and PFMCs were incubated with the following Abs: APC-labeled anti-CD3, FITC-labeled anti-CD3, PB-labeled anti-CD4, PeCy7-labeled anti-CD8 (all these Abs were purchased from BD Bioscience, San Diego, CA, USA), PercpCy5.5-labeled anti-CD14, APC-labeled anti-PD-1, FITC-labeled anti-PD-1, PeCy7-labeled anti-PD-L1, PE-labeled anti-PD-L1 and PE-labeled anti-PD-L2 (all these Abs were purchased from eBioscience, San Diego, CA, USA). Stained samples were fixed with 2% PFA and acquired on a MoFlo XDP flow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed with summit 5.2 (Beckman Coulter).

Spleens were isolated from MRL.Fas^lpr mice at 25 weeks of age, and immune cell composition was analyzed by flow cytometry. Spleen cells were stained with the following monoclonal antibodies in 0.5% BSA/PBS at 4°C for 15 min: FITC-labeled anti-B220, APC-labeled anti-CD3, PE-labeled anti-CD4, FITC-labeled anti-CD8, FITC-labeled anti-CD11c, PE-labeled anti-CD11b, APC-labeled anti-CD138, FITC-labeled anti-IgG1 (BD Biosciences, San Jose, CA, USA), FITC-labeled anti-CD4, and PE-labeled anti-Foxp3 (eBioscience). Data were collected using FACSCalibur and analyzed with CellQuest Pro software (BD Biosciences).