Anti mouse cd4 apc gk1

Tumor-bearing mice were sacrificed on the last day of treatment. Tumor tissues were minced and digested in collagenase IV (Invitrogen, Carlsbad, CA, USA) and Dnase I (Sigma–Aldrich) for 30 min at 37 °C, and passed through a 70 μm cell strainer. After centrifuging, part of the cells were collected and stained with surface markers antibodies anti-mouse CD45 FITC (30-F11, eBioscience), anti-mouse CD3 PerCP-eFluor710 (17A2, eBioscience), anti-mouse CD8α PE (53–6.7, eBioscience) for 30 min at 4 °C to detect CD4⁺ T cells and CD8⁺ T cells.
Another part of the cells 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 permeabilization. Permeabilized cells were then stained with anti-mouse FOXP3 PE-Cy7 (FJK-16s, eBioscience) for 30 min at 4 °C to detect FOXP3⁺ Tregs. The cells were washed and analyzed by a FACS Calibur flow cytometry.

Single cell suspension of mouse spleen and draining lymph node were prepared by gentle mechanical disruption, tumor-infiltrating lymphocytes (TIL) were isolated from tumor tissues. Cells from CT26 xenograft model were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich) and 1 μmol/L ionomycin (Sigma–Aldrich), and cells from B16-OVA xenograft model were stimulated with 10 μg/mL OVA_257–264 peptide in the presence of protein transport inhibitor cocktail (eBioscience) for 4 h. Cells were then stained with surface markers antibodies anti-mouse CD3 PerCP-eFluor710 (17A2, eBioscience), anti-mouse CD8α APC (53–6.7, eBioscience) or anti-mouse CD4 APC (GK 1.5, eBioscience) prior to fixation and permeabilization. Permeabilized cells were then stained with anti-mouse IFN-γ PE (XMG1.2, eBioscience), analyzed by a FACS Calibur flow cytometry.