Rtca sp

Background was measured with 50 µL medium per well in an E-Plate 16 (Roche). Then 3×10³ or 5×10³ cells/well were seeded in 150 µL additional volume. Cell attachment was monitored with RTCA SP (Roche) instrument and the RTCA Software V.1.2 (Roche) every 10 min. After 16–20 hours, melanoma cells were transfected with 100–200 ng/mL 3pRNA or Ctrl RNA and incubated for 10–15 days at 5% CO₂, 37°C. As additional controls, cells were treated with Lipofectamine 2000 only or left untransfected. Six hours post-transfection, 10% FCS was added to Ma-Mel-86c and Ma-Mel-47 cells, the medium was exchanged entirely for UKE-Mel-164a cells. All experiments were carried out as triplicates. Changes in the electric impedance were given as a dimensionless cell index value derived from relative impedance changes corresponding to cellular coverage of the electrode sensors. The cell index was first normalized to the baseline impedance value of the background and then to the time point of transfection.

Real-time cytotoxicity assays were performed as described previously (Peper et al., 2014 (link)). All experiments were performed in DMEM with 10% FCS and 1% penicillin/streptomycin. Background values were determined using 50 µl medium per well. MRC-5 cells, infected or mock-treated, were seeded in 96-well E-plates (ACEA Biosciences; 05232368001) at a concentration of 20,000 cells per well in 50 µl medium. Effector cells were added 48 h after target cells in indicated E:T cell ratios. For peptide loading of MRC-5 cells, synthetic peptides (final concentration 1 µg/ml) were added to target cells 1 h before effector cells. Cell attachment was monitored using the RTCA SP (Roche) instrument and RTCA software version 1.1 (Roche). Impedance measurements were performed every 15 min for up to 140 h. All experiments were performed in triplicates.