Xcelligence instrument

Real-time proliferation assays were carried out using an xCELLigence instrument (ACEA Biosciences). MCF7-BUS cells were maintained in phenol red-free DMEM supplemented with 5% charcoal-stripped FBS and 1% penicillin-streptomycin (starvation medium) for 1 week before the proliferation assay. At the start of the assay, 50-µL starved medium was added to each well of an E-plate 16 (ACEA Biosciences) and left to equilibrate for 30 minutes before taking a background reading. During this time a suspension of the MCF7-BUS cells (100 000 cells/mL) was prepared in starvation medium. A total of 100 µL of the cell suspension was then added to each well and the plate was equilibrated for a further 30 minutes prior to initializing the run on the xCELLigence instrument. Each run consisted of 400 sweeps with a 30-minute time interval. After 24 hours, 50 µL of unsupplemented phenol red-free DMEM containing 1 nM steroid (E₂ or 11OHE₂) was added to each well and proliferation monitored over 140 hours.

The migration potential of MCF7 cells was monitored by a real-time technique using the xCELLigence Instrument (Acea Biosciences, Euroclone) and CIM-16 plates, following manufacturer's instructions. Prior to the analysis, cells were grown in estrogen-free medium for two days and left untreated (mock) or treated with Doxo, TNF⍺ or the combination. 16 hours after the treatments, cells were detached and added to the top chamber in serum-free medium. Migration was detected every 10 minutes for 24 hours. We used 0.5% and 5% FBS as chemo-attractant. Migration and Invasion were also measured by QCM^TM Fluor 24-Well Cell Migration and Cell Invasion kits (Merck-Millipore, Milan, Italy), according to manufacturer's instructions. For wound healing, cells were seeded in 12-well plates and treated with Doxo, TNF⍺ or the combination. After 16 hours a scratch was introduced using a 10 μl pipette tip. Images of the same field were acquired immediately (T0) and after 24 hours (T24) using an automated Zeiss microscope and the AxioVision3.1 software in multidimensional mode with mosaic (3×3) acquisition.

Sensitivity of KB-8-5 cells loaded with tON/asON duplexes to vinblastine was estimated by monitoring cell survival for 120 h in real time mode with an xCELLigence instrument (ACEA Biosciences, Santa Clara, CA, USA). KB-8-5 cells were seeded in 16-well E-plates at a density of 5 × 10⁴ cells per well and incubated overnight. Then, culture medium was replaced with fresh DMEM without FBS and antibiotics, and supplemented with 6 µM tON/asON duplexes, and cells were incubated for 4 h under SC. Next, FBS and antibiotics were added to the concentrations of 10% and 1%, respectively. Cells were cultured for 24 h under SC followed by 300 nM vinblastine addition and incubation for 96 h under SC.

Polyclonal human AHAs and the monoclonal AHA (2095–2) were examined for their ability to restore ADCC activity using a non-invasive gold microelectrode-based cell cytotoxicity assay by the xCELLigence instrument (ACEA Biosciences, San Diego, CA, USA) as described previously [7 (link)]. SKOV3 and SKBR3 cancer cells were used as target cells (T) and human PBMCs, freshly isolated from two healthy donors, were used as effector cells (E) with the E:T ratio at 25:1. The degree of ADCC restoration by AHA coupled with scIgG-P was by comparison to the cells treated with IgG-P (30 nM), or scIgG-P (30 nM), respectively, with or without AHA (60 nM). The ADCC rescuing efficacy of polyclonal human AHAs or monoclonal AHA (2095–2 mAb) was measured by adding scIgG-P alone or in combination with scIgG-T together with a twofold to tenfold excess of AHAs. The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. All experiments were replicated three times (n = 3).