Anti cd3 mab 145 2c11

The cells (2 × 10⁶) were labeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Molecular Probes, Eugene, OR, USA) and activated with 2 mg/mL anti-CD3 mAb (145-2C11, eBiosciences, Inc., San Diego, CA, USA) and 0.5 mg/mL anti-CD28 mAb (BD Pharmingen, San Diego, CA, USA) in complete culture media, RPMI 1640 (ThermoFisher Scientific, Waltham, MA, USA) containing 10% FBS (ThermoFisher Scientific, Waltham, MA, USA). Intracellular esterases cleave the acetate groups, leading to the fluorescent carboxyfluorescein succinimidyl ester (CFSE). Cell division accompanied by CFSE dilution at 72 h after culture was analyzed by flow cytometry. The proportion of CFSE+ cells proliferating in vitro was calculated as below. The number of cells (events) in each cycle (division: n) was divided by 2 raised to power n to calculate the percentage of original precursor cells from which they arose. The sum of original precursors from division 1 to 6 represents the number of precursor cells that proliferated. The percent of CFSE+ divided cells was calculated by (no. of precursors that proliferated_1–6/no. of total precursors_0–6) × 100. Percentage of suppression in comparison to proliferation of control group lymphocytes was indicated as 100 × (percentage of divided cells of control group percentage of divided cells of rapamycin group)/(percentage of divided cells of control group).

Red blood cells were removed from the spleen using red blood cell lysis buffer (Sigma-Aldrich). The splenocytes were stained with 2.5 μM CFSE for 7 min at room temperature using a CellTrace^TM CFSE Cell Proliferation Kit (Invitrogen). An equal volume of FBS was added to splenocytes and incubated for 3 min. After the cells were washed twice, they were cocultured with 4T1-mock or 4T1-NDRG2 cells in RPMI 1640 (Gibco/Invitrogen) medium supplemented with 10% heat-inactivated FBS. To prevent proliferation of 4T1 cells, cancer cells were pre-treated with mitomycin C for 6 h. CFSE-labeled splenocytes were stimulated using 1 $$\mathsf{\mu }\mathrm{g}$$ /mL anti-CD3 mAb (145-2C11, eBioscience) and 1 $$\mathsf{\mu }\mathrm{g}$$ /mL anti-CD28 mAb (37.51, eBioscience) for 3 days. The cells were stained with an antibody against CD8 (J43, eBioscience), and then CFSE⁺ CD8⁺ T cell proliferation was analyzed by flow cytometry.