Percp cy5.5 anti mouse cd8a

The antibodies used to stain TILs were as follows: FITC anti-mouse CD3 (100203; BioLegend, CA, USA), PE anti-mouse CD4 (100407; BioLegend), Percp/Cy5.5 anti-mouse CD8a (100733; BioLegend), and APC anti-mouse Granzyme B (372203; BioLegend). Using BD LSRFortessa flow cytometer to collect samples, and using FlowJo software for data analysis.

Tumors were harvested and mechanically dissociated into fragments within 2 h. Subsequently, the tumor tissue was disrupted to prepare single cells using a tumor isolation kit (Miltenyi Biotec) following the manufacturer’s instructions. The cell suspension was lysed with a 70 μm cell filter to remove red blood cells. Tumor-infiltrating leukocytes were isolated through gradient centrifugation using a 40%/80% Percoll (GE Healthcare) solution. Subsequently, the collected cells were blocked with Fc block (anti-mouse CD16/32, BioLegend) on ice for 30 min. The samples were first stained for surface markers for lymphoid immune populations before intracellular staining. For FoxP3 and intracellular staining, True-Nuclear Transcription Factor Buffer Set 424,401 (BioLegend) was used following the manufacturer’s instructions. The following antibodies and stain kit were purchased from BioLegend: APC-Cy7 anti-mouse CD45, Alexa Fluor 700 Hamster anti-mouse CD3e, FITC anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8a, BV786 anti-mouse CD45R/B220, PE anti-mouse NK-1.1, BV650 anti-mouse IFN-γ, Alexa Fluor 647 anti-mouse Foxp3 and fixable viability stain 510. PE-CYN7 anti-mouse granzyme B was obtained from Thermo Fisher Scientific.