Pe conjugated mouse igg1k

Lymphocytes were collected from 4 ml peripheral whole blood using Ficoll-Paque PLUS. As phorbol-12-myristate-13-acetate (PMA)-Ionomycin is a potent stimulator of IFN-γ and IL-4 in lymphocytes and monensin as a secretion inhibitor can retain cytokines intracellularly after fixation and permeabilization [24 (link)], cells were stimulated with 50 μg/l PMA (ENZO, Farmingdale, NY), l mg/l Ionomycin (ENZO), and 1.7 mg/l monensin (ENZO) for 4 h at 37 °C in a 5% CO₂ humidified atmosphere. Cells cultured with 1.7 mg/l monensin alone were used as an unstimulated control group. After washing twice with PBS, 100 μl cells (1×10⁶ cells/ml) were stained with 10 μl mouse anti-human PE-conjugated anti-PD-1 mAb (BD Bioscience) for 15 min in the dark at room temperature. Subsequently, the cells were treated with the Fix & Perm Reagent (eBioscience) and then incubated with mouse anti-human FITC-conjugated anti-IFN-γ mAb or anti-IL-4 mAb (eBioscience) for 30 min in the dark at room temperature, respectively. PE-conjugated mouse IgG1k (eBioscience) and FITC-conjugated mouse IgG1k (eBioscience) served as isotype controls. Cells were washed twice with PBS and flow cytometric measurement was performed within 4 h.