Pe anti human ifn γ antibody

The density of infiltrating CD4+ T and CD8+ T cells in the tumours and the expression levels of interferon (IFN)-γ were evaluated using flow cytometry. Fresh GC tumour tissue and mice subcutaneous xenograft tissue samples were taken to obtain tumour-infiltrating lymphocytes. Tumour-infiltrating lymphocytes were suspended, and 1 μl antibodies were added into the tube. Antibodies included Fluorescein isothiocyanate (FITC) anti-human CD8 (980908, BioLegend), FITC Mouse Anti-Human CD4 (550628, BD Biosciences), and PE anti-human IFN-γ Antibody (383303 , BioLegend), FITC anti-mouse CD8a Antibody PE (100705, BioLegend), anti-mouse IFN-γ Antibody (505807, BioLegend). After routine operation, the supernatants were collected and flow cytometry was performed.

Tumor‐specific CD8⁺ T cells were generated as described above and cocultured with HLA‐A2⁺ primary kidney tumor cells at an effector/target (E/T) ratio of 10:1 in 48‐well plates for 12 h at 37 °C. Tumor cells were then stained with PI (ST511, Beyotime) and immediately analyzed by flow cytometry. T cells were collected and then treated with protein transport inhibitor (BD) for 6 h at 37 °C followed by fixation and permeabilization using a Fixation and Permeabilization Solution Kit (554714, BD) according to the manufacturer's instructions. Cells were then stained with PE anti‐human IFN‐γ antibody (502508, Biolegend). Samples were analyzed with a Beckman CytoFLEX Flow cytometer (Beckman Coulter, USA), and FlowJo10 software was used to analyze the data.