Xcelligence real time cell analysis instrument

Migration and invasion assays were performed using CIM-plate 16 on xCELLigence Real Time Cell Analysis instrument, from ACEA Biosciences Inc. (San Diego, CA, USA), which allows quantitative kinetic measurement of cell movement from the upper chamber to the lower chamber. Cells were cultured for 24 h until they reach 60–80% confluence followed by 6-h serum starvation [1% fetal bovine serum (FBS)] before they were harvested and adjusted at 30 × 10³/100 μL in serum-free media. For migration and invasion, 160 μL of chemotaxis (10% FBS) was added to the lower chamber, and cells (100 μL) were added to the upper chamber. For invasion, 25 μL of Matrigel was used to coat the upper chamber for 4 h in the 37°C incubator prior to the addition of cells. The cells were allowed to settle for 30 min at room temperature before loading the plate into the instrument, and measurements were taken at 15-min intervals for 24 h.
Data acquisition was stopped after 24 h of measurement, and the average migration or invasion of all the replicates was calculated using the xCELLigence software. Migration or invasion signal was considered positive if it has a mean Cell Index of ≥0.1. Rate of cell migration and invasion was assessed by increases in the slope (1/h) of the curve during 24 h, and a graph displaying change in the slope between the different groups was generated.

The ADCC assay was performed in xCELLigence Real-Time Cell Analysis Instrument (Agilent, xCELLigence RTCA DP), an impedance-based technology that monitors cell proliferation and attachment in real-time. The day before starting the assay, target CHO/mPSMA_mEGFR cells were plated in E-Plate 16 PET (Agilent #300600890), 40,000 cells/well, to allow them to adhere and reach the exponential growth phase. The following day the human CD11b⁺ effector cells were preincubated with indicated IgE/IgG antibodies at 5 μg/ml for 1 h, at 37 °C and 5% CO₂, after which the unbound antibodies were washed, and 80,000 of IgE- or IgG-coated effector cells were added per well. The cell killing was monitored by the instrument.

Electroporated CLDN6-TCR⁺ CD8⁺ T cells (HLA-A*02) were preactivated for 24 hours by coculture with CLDN6-transgenic MDA-MB-231 tumor cells (MDA-MB-231 hCLDN6) endogenously expressing PD-L1. Preactivated CD8⁺ T cells were transferred to MDA-MB-231 hCLDN6 cells that had been seeded (2 × 10⁴ cells/well) on the previous day in an xCELLigence E-plate (1 × 10⁵ T cells/well; Agilent). Cells were incubated with GEN1046 or control antibodies (0.2 µg/mL) for 120 hours with impedance measured at 2-hour intervals in an xCELLigence Real-Time Cell Analysis Instrument (Agilent). Impedance measurement data were normalized to the time of coculture start for each treatment condition, and data were expressed relative to tumor cells cultured alone (set to 100%). After 48 hours of incubation, supernatants were removed from replicate wells not subjected to impedance measurement for analysis of intracellular GZMB and CD107a. Fresh media containing Brefeldin A (BD Biosciences; 555029) were added to the cells, followed by incubation for 4 hours and flow cytometry.