Non-radioactive killer cell cytotoxicity assay

Abstract

Natural killer cells are lymphocytes that act as the first line of defense against tumors. Natural killer cells do not require previous exposure to target cells for activity. This allows them to control abnormal cell growth prior to the development of an Immune response. These unique immune system cells are able to kill target cells directly, or through antibody dependent cell cytotoxicity. The chromium release assay is the most common procedure used to evaluate natural killer cell lytic activity. This assay requires the use of radioactivity, and thus is expensive. Also, problems with biosafety and waste disposal make this a difficult procedure to establish in the laboratory. Many cells are also resistant to labeling with chromium (Korzeniewski and Callewart, 1983). Lactate dehydrogenase (LDH), a cytosolic enzyme found in most cells, is released when a cell is lysed. Using this information it is possible to study and measure the cytotoxic activities of natural killer cells against different tumor cell lines. The LDH assay used in this study is a non-radioactive, colorimetric alternative to the sensitive chromium release assay. This study examines several tumor cell lines to determine if any contained high levels of LDH and had low spontaneous release of the enzyme. In this study using the non-radioactive cytotoxicity assay, it was observed that, the cell line MPCII had a 3.9 fold higher total LDH release when compared to spontaneous LDH release. The cell line P815 had a 1.9 fold higher total LDH relsease when compared to spontaneous LDH release. The cell line YAC-1 had no detectable LDH release with addition of lysis buffer, suggesting insufficient levels of enzyme.Nulli-SCC had 39 fold higher total LDH release. Cell lines found to be useful using this assay were MPCII, P815 and NULLI-SCC. These cell lines were plated with natural killer cells and % cytotoxicity was measured. These studies demonstrate that the 96 TM cytotoxicity assay may be effectively used to measure natural killer cell mediated cytotoxicity using the MPCII, NULLI-SCC and P815 target cell lines.