Tumor Inoculation and T-cell Isolation

ZsGreen tagged BTSC#233 cell lines were cultured as previously described by us^{37 ,72 (link)}. Post trypsinization, a centrifugation step was performed, following which the cells were harvested and suspended in MEM media at a concentration of 20,000 cells/µl. inoculate Cultured tissue sections were inoculated with 1 µl of tumor cells using a 10 µl Hamilton syringe, and further cultured as required. Matched peripheral blood samples were collected from the cortical tissue donors. Peripheral T-cells were isolated using the same MACSxpress^® Whole Blood Pan T-Cell Isolation Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) and erythrocytes were eliminated from the suspension using ACK-lysis buffer (Thermo Fisher Scientific, Carlsbad, USA). T-cells were then fluorescently tagged using the CellTrace^TM Far Red Cell Proliferation kit (C34564, Thermo Fisher Scientific, Carlsbad, USA) prior to inoculation. Endogenous IL-10 receptor expressed on T-cells were blocked using anti-IL10 neutralizing antibody (ab34843, Abcam, Cambridge, UK) at a concentration of 5 µg/ml for 1 h at 37 °C. Both naïve and neutralized T-cells (40,000 cells/µl) were inoculated into tissue sections and cultured for 48 h.