Anti mouse cd8 apc 53e6

Tumour-bearing mice were sacrificed on the last treatment day. Tumour tissues were minced and digested in collagenase IV (Invitrogen, Carlsbad, CA, USA) and Dnase I (Sigmae-Aldrich) for 30 min at 37 °C, and passed through a 70-mm cell strainer. Part of the cells were collected and stained with surface markers antibodies after centrifuging, anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD3 PerCP-eFluor 710 (17A2, eBioscience), and anti-mouse CD8 APC (53e6.7, eBioscience) for 30 min at 4 °C to detect CD4⁺T cells and CD8⁺ T cells.
Another cell part was stained with surface markers antibodies anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD4 APC (GK1.5, eBioscience), and anti-mouse CD25 PE (PC61.5, eBioscience) prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-mouse FOXP3PE-Cy7 (FJK-16 s, eBioscience) for 30 min at 4 °C to detect FOXP3⁺ Tregs. The cells were washed and analysed by a FACS Calibur flow cytometry.

Single cell mouse spleen and draining lymph node suspension were prepared by gentle mechanical disruption. Tumour-infiltrating lymphocytes were isolated from tumour tissues. Cells from CT26 xenograft model were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma eAldrich) and 1 μmol/L ionomycin (Sigma eAldrich) for 4 h. Cells were then stained with surface markers antibodies anti-mouse CD3 PerCP-eFluor710 (17A2, eBioscience), anti-mouse CD8 APC (53e6.7, eBioscience), or anti-mouse CD4 APC (GK1.5, eBioscience) prior to fixation and permeabilisation. Permeabilized cells were then stained with anti-mouse IFN-γ PE (XMG1.2, eBioscience), analysed by FACS Calibur flow cytometry.