Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells. Cytotoxicity Assay Kit (CFSE, 7-AAD) (ab270780) allows you to assess cytolytic activity in cell culture.

Older methods to assess NK cytolytic activity include measuring the release of lactate dehydrogenase, and more commonly, the release of radioactive ⁵¹Cr from lysed target cells. Unfortunately, these techniques have several drawbacks. Traditional enzyme-release assays are often skewed by the large number of necrotic effector cells. Problems associated with ⁵¹Cr release methods include high spontaneous leakage resulting in high backgrounds, high cost, short half-life, and the health risks due to exposure to radioactive material.

Flow cytometric assays have been developed to overcome some of the difficulties associated with older assays like lactate dehydrogenase and ⁵¹Cr release assays. One such early version involved the detection of NK cytotoxicity activity by staining target cells with the green fluorescent dye, F-18, in combination with the DNA intercalating dye, propidium iodide . Since then, a red fluorescent membrane dye, PKH-26, has been used in preference to F-18, and in combination with the viability probe, TO-PRO-3 iodide4-7. However, despite correlations of greater than 95% when compared with the ⁵¹Cr release assay, the PKH-26 method is problematic. It is difficult to use at a constant concentration, leading to unreliable staining, and the staining procedure requires multiple steps, often decreasing the viability of the target cells.

Cytotoxicity Assay Kit (CFSE, 7-AAD) includes two fluorescent reagents: CFSE and 7-AAD. CFSE, a green fluorescing cellular stain, is utilized to identify the target cell population. The unstained effector cells are added and incubated with the target cells. 7-AAD, a red fluorescing live/dead stain, is then added to stain all necrotic cells red by binding to the DNA of membrane-compromised cells. The target cell and effector cell populations can then be easily distinguished, and flow cytometry easily identifies live and necrotic cells.