Cell trace red

Four hours after plasma treatment, tumor cell culture supernatants were collected, centrifuged at 1000× g for 5 min to discard residual cells and debris, and stored at −20 °C until use. For experiments, 80 µL of this supernatant was added to wells of a flat-bottom 96-well plate (Eppendorf, Hamburg, Germany). To each well, 20 µL of a cell suspension containing 1 × 10⁴ of either THP-1 or HL-60 cells was added. The myeloid cells were incubated for up to 96 h. The Eppendorf 96-well plates have an outer rim that was filled with deionized water to prevent excessive evaporation from the outer wells, as observed with extensive culture durations. For the co-culture of prostate cancer and myeloid cells, the 96-well plates were coated with 0.01% poly-l-lysine. 2.5 × 10⁴ (LNCaP) or 1.25 × 10⁴ (PC3) in 100 µL of cell culture medium cells were labeled with trace violet (Thermo Fisher Scientific, Dreieich, Germany) before being added to each well. The cells were exposed to the gas plasma and incubated for another 30 min in the incubator. Subsequently, 2.5 × 10⁴ or 1.25 × 10⁴ THP-1 or HL-60 cells were labeled with cell trace red (Thermo Fisher Scientific, Dreieich, Germany) and added to the tumor cells together with sytox green (Thermo Fisher Scientific, Dreieich, Germany) for the identification of terminally dead cells.