Cytotoxicity assay by Real-Time Cell Analyzer

Cytotoxic effector activity of short-term expanded γδ T-cell lines against U251MG and BxPC3 target cells was determined by Real-Time Cell Analyzer (RTCA, X-Celligence, ACEA, San Diego, CA, USA) which measures the decrease of impedance of adherent tumor cells over extended time periods as a correlate of cell lysis. RTCA was performed as previously described.³⁹ Briefly, 10⁴ tumor cells in complete medium were added to each well of the micro-E plates. After overnight incubation, γδ effector T cells were added at an effector/target ratio of 10:1 or 2:1 with or without STING ligands and/or HMBPP as a positive control to enhance TCR-dependent lysis. The impedance of the cells was recorded via electronic sensors on the bottom of the 96-well micro-E-plate every 3 min for up to 48 h. Results were analyzed with RTCA software (version 2.0.0.1301, ACEA Biosciences Inc.) and normalized as described.³⁹ Results of several experiments with different γδ T-cell lines are summarized as percentage of cell death induced by γδ T-cells at various time points in relation to the corresponding tumor cell index in medium and in Triton-X100 (maximal lysis). Time point zero was defined as first measurement after addition of γδ T cells.