Cd3 mab

Lymphocytes (1 × 10⁷ cells/ml) in ascites were incubated with 2 μM Carboxyfluorescein diacetate, succinimdyl ester (CFSE, BioLegend) at 37°C for 20 min. After washed with PBS, cells were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% inactivated fetal calf serum (Gibco) and 1% penicillin/streptomycin (Hyclone) and subsequently stimulated with CD3 mAb (2 μg/ml; BioLegend) and CD28 mAb (1 μg/ml; BioLegend) for 72 h. Unstimulated PBMC were included as control. Cells were collected, and CFSE signal was measured by flow cytometry.
Alternatively, lymphocytes in ascites were directly stimulated with CD3 mAb (2 μg/ml; BioLegend) and CD28 mAb (1 μg/ml; BioLegend) for 72 h. Cells were collected, stained with Ki67, and measured by flow cytometry.

PBMCs (5×10⁵ cells/mL) were incubated with 2 mmol/L Carboxyfluorescein diacetate succinimdyl ester (CFSE, BioLegend) at 37°C for 20 min. After washed with precooled PBS, cells were cultured in RPMI 1640 medium (Hyclone) supplemented with 10% inactivated fetal calf serum (Gibco) and 1% penicillin/streptomycin (Hyclone) and subsequently stimulated with CD3 mAb (2 μg/mL; BioLegend) and CD28 mAb (1 μg/mL; BioLegend) for 72 h. Unstimulated cells were included as control. Cells were collected, and the CFSE signal was measured by flow cytometry.