Pe anti human pd 1

The peripheral blood mononuclear cells (PBMC) of CRC patients were isolated by centrifugation with erythrocytes lysate and were used to analyze PD1⁺CD8⁺T cells and CD3^-CD56⁺CD16⁺NK cells by flow cytometry. The PBMC were stained for 30min on ice using the following antibodies: FITC anti-human CD8 (344704, Biolegend), PE anti-human PD1(367404, Biolegend), APC anti-human CD3 (300312, Biolegend), PE anti-human CD56 (985902, Biolegend), and PerCP anti-human CD16 (302030, Biolegend). Stained cell suspensions were analyzed using the BD flow cytometer (BD Accuri C6 Plus), and data analysis was performed using FlowJo_v10.8.1.

The following cell surface fluorochrome-conjugated monoclonal antibodies were used to detect T cells phenotype: FITC anti-human CD4, PE/Cy7 anti-human CD8, PE anti-human CD3 (Biolegend, San Diego, CA, USA) and APC anti-human NKG2D (eBioscience). T cell activity was determined by staining the surface APC anti-human CD69, intracellular PE anti-human IFN-γ (BD Biosciences, San Diego, CA, USA), PE anti-human GzmB (eBioscience, San Diego, CA, USA) and PE anti-human Bcl-2 (Biolegend) antibodies. PE anti-human PD-1 and APC anti-human Tim-3 were purchased from Biolegend. FITC anti-human CD45RA, Percp-cy5.5 anti-human CCR7 and PE anti-human CD45RO were purchased from Biolegend to determine T cell subsets. APC Annexin-V and 7-Aminoactinomycin D (7-ADD) from BD Biosciences were used for apoptosis staining. APC mouse IgG1, κ isotype, PE mouse IgG1 isotype, PE/Cy7 mouse IgG2a, κ isotype, FITC mouse IgG1 isotype, PE/Cy7 mouse IgG1, κ isotype (Biolegend) were used as controls. Flow cytometry was performed on the BD LSRFortessa flow cytometer, and data were analyzed using FlowJo Version 10 software.