Quantitative Cytotoxicity Assay for Myeloma

The quantitative cytotoxic assay for measuring LDH was as described previously [21] (link). We used the CytoTox 96 non-radioactive cytotoxicity assay (Promega, USA) to analyze the direct killing effect of activated lymphocytes against patient autologous primary myeloma cells, according to the manufacturer's instructions. The patients’ autologous primary myeloma cells were pre-stained without or with 2 µg of anti-human HLA-A, B, or C antibody (clone: W6/32, Biolegend, USA) per 10⁶ cells in 100 µL of RPMI media for 20 min, and then washed via centrifugation. The patient primary myeloma cells were co-cultured with activated lymphocytes at a 1:2 ratio in a Costar 96-well plate (Corning, USA) for 6 h at 37°C with 5% CO₂. Then the supernatants were collected to measure the concentration of LDH, which is the cytosolic enzyme that is released upon cell lysis. The percentage of cell lysis was calculated according to the manufacturer's instructions.