Anti human pd 1 bb515

Collected peripheral blood samples were analyzed using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque centrifugation, then stained by the following antibodies: anti-human CD3-APC-H7 (BD biosciences, clone: SK7), anti-human TCR γδ-BV421 (BD biosciences, clone: 11F2), anti-human PD-1-BB515 (BD biosciences, clone: EH12.1), anti-human NKP46-BV510 (BD biosciences, clone: 9E2/NKP46), anti-human NKP30-Alexa Fluor^®647 (BD biosciences, clone: P30-15), anti-human NKG2D-PE-Cy™7 (BD biosciences, clone: 1D11), anti-human TCR Vδ2-PE (BD biosciences, clone: B6), and anti-human TCR Vδ1-PerCP-Vio700 (Miltenyi Biotec, clone: REA173). Data was analyzed using FlowJo 10.1 software (Tree Star Inc., Ashland, OR, USA).

For binding ability of CHI3L1 to receptor, NSCLC cell line H1299 was harvested and thoroughly mixed with His-tagged rCHI3L1 or nCHI3L1 Ab for 1 h. Subsequently, cells were incubated with FITC-labeled 6x-His Tag antibody at 4 °C for 1 h and analyzed by FACS. To determine PD-1 and TIM-3 presentation on CD8⁺ T cells, cells were stained using anti-human PD-1-BB515 (BD Bioscience) and TIM-3-BV421 (BD Bioscience) antibodies. After cell surface staining, intracellular CD107a was stained with CD107a-PE (BD Bioscience) antibodies to detect T cell activities.
Allograft tumor tissues were digested with 0.1 mg/mL collagenase (Sigma-Aldrich) and 1 mg/ml dispase II (Sigma-Aldrich) at 37 ℃ for 30 min and meshed. Resulting single-cell suspension were stained with CD4, CD8, CD11b, CD25, CD86, CD206, CTLA4 and Foxp3 antibody (BD Bioscience). The data were recorded by CytoFLEX (Beckman Coulter). The detailed antibodies conditions are listed in Table S2.