Measuring murine natural killer cell cytotoxicity using a lactate dehydrogenase assay /

Abstract

Natural killer cells are among the first line of immune defense against virus infected or tumor cells. Natural killer cells possess the ability to lyse target cells by releasing enzymes that perforate the target cell membrane. The lactate dehydrogenase assay measures the amount oflactate dehydrogenase, an intracellular enzyme, released by the target cell when it is lysed. When the LDH interacts with the substrate mix, the conversion of a tetrazolium salt (!NT) into a formazan product produces a colorimetric reaction that can be measured using a standard microplate reader. The amount oflactate dehydrogenase released is an indication ofhow much the natural killer cells are killing the target cells. LI21O, P8I5, Yac-I, and MPC 11 cells lines were tested for their usefulness as target cells when using this assay and murine spleen cells as effector cells. The results indicate that the MPC 11 (45% cytotoxicity) and Yac-I (19% cytotoxicity) cell lines are targeted by murine NK cells and are useful positive controls for measuring murine NK cell cytotoxicity with the LDH assay. In our study, cytotoxicity was also observed with the LI210 cells, however, as these results are in contrast with previous reports, additional tests are required to confirm these results. The data also indicates that the P8I5 cell line is not targeted for lysis by murine NK cells. This cell line would be useful as a negative control in murine NK cell cytoxicity studies. While these studies have shown that the LDH assay can be used to measure murine NK cell function, the assay is not effective at all effector cell concentrations. The cytotoxicity at the higher E:T ratios was masked by the high amount of murine NK cell spontaneous LDH release. The high levels of spontaneous LDH release requires that careful titration of effector cells be performed when measuring murine NK cell cytotoxicity using the LDH assay.