Rtca software 2

Proliferation and migration analyses were performed using a xCELLigence Real-Time Cell Analyzer (RTCA) (ACEA Biosciences, San Diego, CA, USA). For measurement of proliferation dynamics, cells were seeded in 200 µL of growth media at a density of 5000 cells/well into an E-plate 16 (ACEA Biosciences, USA). Proliferation curves were evaluated over the course of 72 h with measurements taken every 15 min. Doubling time was analyzed from 8 to 43 h using RTCA software 2.1.0 (ACEA Biosciences, USA). For measurement of migration dynamics, 15,000 cells/mL density were seeded in 100 µL of serum-free media in upper chambers of CIM-plates 16 (ACEA Biosciences). The lower chamber was filled with 160 µL of growth media as a chemoattractant. Cell migration was evaluated over the course of 24 h with measurements taken every of 15 min. Slopes of migration curves were analyzed from 15 to 23 h using RTCA software 2.1.0 (ACEA Biosciences, USA).

Real-time monitoring of cell proliferation was performed using the xCelligence RTCA DP system (ACEA Biosciences). Background impedance was measured 30 min after adding 100 μl of culture medium to E-plates 16 PET (ACEA Biosciences). Next, either 2,500 dermal Fb or 3,000 HPASMC per 100 μl of culture medium per well were seeded. After 30 min incubation at room temperature, plates were placed into the xCelligence system. The relative change in electrical impedance termed the Cell Index (CI) was measured. Fb and HPASMC were transfected with negative control or ASO targeting OTUD6B-AS1 26 h or 50 h after seeding, respectively. CI was monitored every 5 min from 0 h to 13 h, every 15 min from 13 h to 25 h and every 30 min from 25 h until CI reached plateau. CI was normalized at the time of transfection for every experiment. The RTCA software 2.0 (ACEA Biosciences) was used to calculate the slope of the CI curve as measure of cell proliferation.

CTL lysis of PDA30364/OVA cells was assessed in vitro using the impedance-based xCELLigence Real-Time Cell Analyzer System (RTCA) (ACEA Biosciences, San Diego, USA). Eighteen h after irradiation, tumor cells were seeded into an E-Plate 96 (ACEA Biosciences) at a density of 7.2 × 10³ cells/well and rested overnight at 37 °C. Next day, CTLs were added at an effector/tumor cell ratio of 2.5:1. PD-L1-specific antibody (20 µg/ml) (Bio X Cell, Inc., West Lebanon, USA) was added 3 h prior to CTL addition and at the time point the co-culture was started, resulting in a final concentration of 10 μg/ml αPD-L1 antibody. The cell index (CI), representing the relative impedance as a measure for the number of adherent cells was determined every 5 min for at least 24 h. CI values were normalized to the time point of CTL addition using the RTCA Software 2.0 (ACEA Biosciences). Percentage cytolysis was calculated according to the formula: Cytolysis [%] = ((CI_wo.CTLs − CI_w.CTLs)/CI_wo.CTLs) × 100. Standard deviation (SD) of mean CI values was calculated using error propagation formulas established by the Biostatistics Department of the DKFZ. Specificity of the CTL line was controlled using parental PDA30364 cells in comparison to PDA30364/OVA cells as described previously^{25 (link)}. “Kill-Time-50” (KT50) was defined as time span between CTL addition and eradication of 50% of PDA30364/OVA cells.