The most commonly used method to measure CMC/ADCC is a radioactive chrominum-51 (⁵¹Cr) release assays (2). There are several disadvantages with this assay: it is expensive, difficult to load certain cell types, expensive to dispose of due to strict environmental regulations, and has high background from spontaneous release of ⁵¹Cr. With the use of flow cytometry, it is now possible to eliminate the need for radioactive material and increase 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 (3,4,5,6).

A cell tracking dye CFSE ( 7,8,9) is utilized to label the target cell population. After the assay has run its experimental protocol, 7AAD (live/dead) (10,11) 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 postitve cells. % cytotoxicity is calculated by the following equation (see experimental example below):

7AAD positive (upper right quadrant)=R1/ 7AAD Postitve (upper right quadrant)= R1 + 7AAD negative (lower right quadrant)=R2 x100

Tetanus toxoid provides efficient T-cell help for the induction of HA-1^H cytotoxic T cells - Britta EizVesper, Peter A. Horn, Claudia Daubert, Barbara Khattab, and Rainer Blasczyk - Transplantation and Cellular Engineering 2006