Anti cd8 mabs

Cytoxicity of CTLs of immunized mice was determined by quantitative measurements of the release of lactic dehydrogenase (LDH). The mouse melanoma B16 cells (H-2^b), colon carcinoma CT26 cells (H-2^d) or 2E8 cells (mBAP31-depleted B16 cells) were pulsed with or without corresponding peptides (2 μg/mL) and used as target cells. CD8⁺ cells, isolated from splenocytes of immunized C57BL/6 mice by magnetic beads conjugated with anti-CD8 mAbs (BD pharmingen), served as effector cells. CTL assays were performed with effector cells (E) and target cells (T) (1 × 10⁴ cells/well) mixed together at ratios of 50:1, 25:1 or 12.5:1 in a final volume of 100 μl. After 4 h incubation at 37°C, 50 μl of the cultured supernatants was collected to assess the amount of LDH release using Non-Radioactive Cytotoxicity Assay kits (Promega). The percentage of target cell lysis was calculated from the following equation: 100 × (A-B)/(C-D), where A is the reading value of experimental signal, B is spontaneous background signal value of the effector cells, C is maximum signal value from target cells, and D is spontaneous background signal value of the target cells.