Chapter 4 │ Page 126 2.5. NTP Treatment A microsecond-pulsed dielectric barrier discharge (DBD) system, described in our previous work [18, 32, 56, 57], was used for the experimental part of this work. The setup comprises a microsecond-pulsed power supply (Megaimpulse Ltd, Russia), with a round-bottom dielectrically covered electrode, providing an electrically and thermally stable plasma [18]. Operation parameters can be found in Table 1. Table 1. Experimental parameters for DBD-NTP application Electrical and Operating Parameters Pulse voltage amplitude 30 kV Pulse rise time 1-1.5 µs Pulse width 2 µs Pulse frequency 200 and 500 Hz Treatment time 10 s Application distance 1 mm For all experiments, this microsecond-pulsed DBD system was used to treat HNSCC cells in 24-well plates when a confluence of 80% was reached, one day after experimental seeding. Cell culture medium was removed immediately prior to NTP application and the 24-well DBD probe was lowered into the well to treatment distance (1 mm), using a z-positioner. Right after treatment, cells were overlayed with fresh culture medium. Plates were incubated for a day at 37°C and 5% CO2 for 24h post-treatment analysis, or processed right after treatment for immediate (0h) analysis. 2.6. Flow Cytometry Analysis Of NK ligands On HNSCC Tumor Cells The NK ligands included in this study were measured individually via a dual staining of a viability stain (LIVE/DEAD™ Fixable Near-IR, APC-Cy7), and a monoclonal antibody against the target ligand (PE anti-HLA-C, anti-HLA-E, anti-CD155, antiCD122, anti-CD73, or anti-MICA/B). Ligand expression was measured to establish a clear temporal separation between immediate chemical and later biological
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