Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays

Abstract In vitro antibody-dependent cell-mediated cytotoxicity(ADCC) assaysare routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cellsfollowing interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g. , lactate dehydrogenase, or measurement of released exogenous labels, e.g. , 51 Cr, europium or calcein. ADCC assaysbased on the detection of exogenous labelsreleased from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeledimmediately prior to assay, which inadvertently introduces additional assayvariations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre- labeledtarget cells for ADCC assays. Thaw-and-use target cells in our experiments were pre- labeledwith the fluorescent dye calceinAM, cryopreserved in single-use aliquots and used directly in assaysafter thawing. Upon thaw, the pre- labeledcells displayed viability and labelretention comparable to freshly labeledcells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre- labeledtarget cells in ADCC assayscan help to alleviate both cell culture and dye labelingderived variability, increase the flexibility of assayscheduling and improve assayconsistency and robustness.