Analyzing Anti-Tumor Cytokine Secretion

The secretion of anti-tumor cytokines from SN-derived T cells with SIGLEC15 knockdown was analyzed using LEGENDplex^TM Multi-Analyte Flow Assay Kit (BioLegend, San Diego, CA, United States) as described previously (Wallrapp et al., 2017 (link)). Single-cell suspension prepared from the SN of five CRC patients were cultured in serum-free medium and transfected with SIGLEC15-specific siRNA or control siRNA for 72 h. The transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin overnight, and the production of interleukin-2 (IL-2), Interferon-γ (IFN-γ), and tumor necrosis factor alpha (TNF-α) in the supernatant was determined by flow cytometry.