Flow Cytometry Cytotoxicity Assay

The immune system is capable of recognizing and destroying target cells, such as tumor or intracellular pathogen infected cells, through a process known as cell mediated cytotoxicity (CMC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Evaluation of this CMC/ADCC activity is one of the most important immunoassays to monitor the status of the immune system. The most commonly used method to measure CMC/ADCC is a radioactive chromium-51 (51Cr) release assays. There are several disadvantages with this assay in that it is expensive, difficult to load certain cell types, strict environmental regulations makes waste disposable expensive and spontaneous release of 51Cr results in high background. With the use of flow cytometry, it is now possible to eliminate the need for radioactive material and increased the ability to quantify cytolytic activity on a single cell bases. Various groups have demonstrated that measuring CMC/ADCC activity by flow cytometry has a strong (95%) correlation with the traditional 51Cr release assay. A cell tracking dye CFSE analog is utilized to label the target cell population and thus separating them form the effecter cell population. After the assay has run its experimental protocol, 7AAD (live/dead) is added to measure cell death. 7AAD only enters membrane compromised cells and binds to DNA. Flow cytometry is utilized to gate on the target cells and measure 7AAD negative vs. 7AAD positive cells.