Cytolytic Assays

This chapter provides an overview of cytolytic assays. Cytolytic pathways, although capable of mediating the extrinsic pathway, are primarily induced through perforin-mediated delivery of granzymes. The earliest means to assess cytotoxicity were those related to trypan blue exclusion of nonviable cells and projection of a microscopic field onto a screen for direct visual counting. These were supplanted by a variety of radioactive and fluorescent strategies to facilitate higher throughput and accuracy. The chromium release microcytotoxicity assay (CRA) is the standard and most frequently used assay for assessment of cell-mediated cytotoxicity in vitro. "Europium release assay" is sensitive and specific and performs well with nonadherent tumor cell targets. Additional advantages are the reduced health risk and radioactive waste and lower cost per assay. The "MTT (3-(4,5-dimethylthiazol.2-YL)-2,5-dipheyltetrazolium bromide) assay" relies on the cellular reduction of tetrazolium salts to their formazan crystals. The advantages of the MTT assay include ease and rapidity of performance, reproducibility of the results, and observed clinical correlation between in vitro and in vivo testing. Because of the disadvantages of CRAs, a number of flow cytometry based killing assays are developed. In most of these assays, target cells are stained with individual markers and after incubation with effector cells, target cell lysis is measured.