Chapter 3 │ Page 73 Table 1. Experimental parameters for NTP application with the DBD system Electrical and Operating Parameters Pulse voltage amplitude 30 kV Pulse rise time 1-1.5 µs Pulse width 2 µs Pulse frequency 200 Hz, 500 HZ, 1000 HZ Treatment time 10 s, 30 s Application distance 1 mm For all spheroid experiments, three-day old HNSCC tumors were transferred to a 24-well plate in the presence of a minimal amount of culture medium, immediately prior to the NTP application. A treatment distance of 1 mm from the 24-well DBD probe was maintained using a z-positioner. Right after treatment, tumor spheroids were transferred back to the 96-well ULA plates, overlaid with fresh culture medium, and incubated at 37°C and 5% CO2 until further analysis. 2.3.2. Cisplatin treatment Cisplatin (CDDP) (S1166, Selleck Chemicals) was administered, immediately after NTP exposure, using the automated D300e Digital Dispenser (399177, Tecan®), enabling automated and precise drug delivery. CDDP stock concentrations were further diluted in a 1:1 ratio in an aqueous medium containing Tween-20 (30%). CDDP sensitivity is cell line-specific and based on an 7-point titration experiment, the working concentrations of CDDP were defined as follows: (1) CDDPlow 1.71 μM and CDDPhigh 5.85 μM for Cal27, (2) CDDPlow 0.925 μM and CDDPhigh 3.16 μM for SCC22B, and (3) CDDPlow 5.85 μM and CDDPhigh 20 μM for SCC61. 2.4. Cell Viability Analysis HNSCC spheroid viability was determined 24 and 48 hours after treatment with the NTP-CDDP combination or the respective monotherapies, with the CellTiter-Glo® 3D Work cell viability assay (G9682, Promega Benelux B.V.). Spheroids were
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