Xcelligence real time cell analysis

To generate SIINFEKL-specific CTLs, splenocytes from OT-1 mice were cultured in the presence of SIINFEKL (2 µg/ml) with daily 2:1 splitting. After 72 hours in culture, >80% of remaining cells are CD8+Vα2+ cells (data not shown). Following the addition of OT-1 CTL effectors to 1×10⁴ target cells plated in a 96-well E-Plate (ACEA Biosciences), alteration of impedance was acquired using the xCELLigence Real-Time Cell Analysis (RTCA) platform per manufacturer recommendations. Triton X-100 (0.2%) was added to some wells to verify complete loss of cell index with total cell lysis, and CTLs alone were plated up to 1×10⁶/well to verify that they do not contribute to gain of impedance (data not shown). Percent loss of cell index for a given time point was calculated as: 1-(experimental cell index/control cell index).

For cytotoxicity experiments, cells were plated on T-75 flasks and grown until they reached at 70-80% confluency. Cell proliferation was monitored in xCELLigence Real-Time Cell Analysis (RTCA) (ACEA Biosciences Inc., San Diego, CA, USA) system using E-Plate 96 or E-Plate 16 (ACEA Biosciences Inc., San Diego, CA, USA) that are integrated with gold microelectrode arrays in the bottom of each well. 100 μl of culture media was added into the plates, and the plates were inserted into the station for background measurement. Cells were then seeded into the wells (5,000 cells/well in 100 μl) and left to attach overnight. The medium was then removed and replaced with 100 μl of fresh medium containing indicated concentrations of compounds. Impedance was measured every 10 minutes during 48 hr and is represented as cell index.

MDA-MB-468 cells were maintained in DMEM (Corning) supplemented with 10% FBS (Corning) and 1% penicillin/streptomycin (ThermoFisher). Healthy human T cells were obtained from the Human Immunology Core (University of Pennsylvania) and expanded as previously described.^{26 (link)} Briefly, CD4 and CD8 T cells were incubated 1:1 and stimulated with CD3/CD28 Dynabeads (Gibco). Human IL-2 (Gibco) was maintained at a concentration of 50 IU/mL for 10 days. The Dynabeads were removed after 7 days of culturing, and the cells were maintained at 0.5–1 M/mL an additional 7 days. The cells were frozen down using a 1:1 mixture of X-VIVO media (Lonza) and 10% DMSO in FBS and allowed to rest in RPMI media (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin 24 hours before cytolysis assays.
10,000 tumor cells were seeded per well 24 hours prior to adding BsAb treatments and T cells at an E:T ratio of 10:1. Controls included 3-fold serial dilutions starting at 100 nM or 10 nM of the monoclonal antibodies alone (Cetuximab and OKT3 separately) and an equimolar mixture of Cetuximab and OKT3. Tumor cytolysis was tracked up to 72 hrs post-treatment using xCelligence Real-Time Cell Analysis (ACEA Biosciences). Data were analyzed and plotted with GraphPad Prism8.