Cd3ε 145 2c11

For analysis of apoptotic cell death, cells were washed with PBS and resuspended in Annexin V binding buffer, and stained with Annexin V-FITC and propidium iodide (PI) at room temperature for 15 min. For immunophenotypic analysis, single-cell suspensions were prepared and stained with indicated antibodies at 4°C for 30 min. Fluorochrome-conjugated antibodies used were as follows: CD3ε (145-2C11, BioLegend), CD4 (RM4-5, BioLegend), CD8a (53-6.7, BD Pharmingen), CD25 (PC61, BD Horizon) and CD44 (IM7, BD Pharmingen). For intracellular staining of Myc, cells were first stained by specific surface antibodies and then fixed and permeabilized with BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s instructions. Cells were stained with a mouse anti-Myc antibody (9E10, Santa Cruz) and then an Alexa Fluor 647-conjugated anti-mouse IgG antibody (Cell Signaling Technology). Above all, acquisition was performed on an Accuri C6 (BD Biosciences) and flow cytometric sorting was conducted on a FACS Aria (BD Biosciences). Live cells were gated based on FSC and SSC characteristics. All acquired data were analyzed with FlowJo software (TreeStar).