Pe cy5 labeled anti cd8

Tumors isolated from the mice were digested mechanically to obtain single-cell suspensions. The cell suspensions were surface stained with PE-Cy7-labeled anti-CD3 (eBioscience, San Diego, CA, USA), FITClabeled anti-CD4 (eBioscience, San Diego, CA, USA) and PE-Cy5-labeled anti-CD8 (eBioscience, San Diego, CA, USA) monoclonal antibodies, and then treated with Fixation/Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ, USA). Then, the cells were stained intracellularly with PE-labeled anti-FoxP3 (eBioscience, San Diego, CA, USA) and Paci c Blue-labeled anti-Granzyme B monoclonal antibodies (Biolegend, San Diego, CA, USA). The stained cells were analyzed with CytoFLEX LX ow cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using CytExpert software (Beckman Coulter, Brea, CA, USA).