Cell-Mediated Cytotoxicity Assays

Methods measuring cell-mediated cytotoxicity can be divided in two based on whether radioactive material is used or not. NK cellmediated cytotoxicity is routinely measured with a short-term assay by utilizing radioactive chromium (51Cr) marking the target cells. This method has several advantages. It is highly sensitive, easy to execute, has low spontaneous release, and utilizes a marker that is nontoxic to the cells. The drawbacks of the method include the short half-life of the label, and the necessity for handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/ enzymatic assays are highly sensitive and easy to use but unfortunately work well only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of these dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false results resulting in decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.