Rpmi 1640 medium

Freshly isolated PBMCs were seeded on 48-well plates (Corning, Burlington, ON, Canada) in RPMI1640 medium (PanBiotech Aidenbach, Germany,) supplemented with 10% FBS (PanBiotech, Aidenbach, Germany). Next, PBMCs were stimulated with LPS 1 µg/mL (Invivogene, San Diego, CA, USA) or phorbol 12-myristate 13-acetate (PMA) and ionomycin (2 µg/mL) (Invitrogen, Bleiswijk, Netherlands), or left unstimulated (vehicle control) in the presence or absence of monocarbonyl analogs of CUR 187.5, 93.75, and 46.88 μg/mL for compounds 3, 6, 11, and curcumin and 5.86, 2.93, and 1.46 μg/mL for compound 12 and 2.93, 1.46, and 0.73 μg/mL for naproxen. After a 24 h stimulation, the supernatant was collected and biobanked in −80 °C for further analysis.

The DLBCL cell line U2932 was kindly provided by the Dr. Senckenberg Institute of Pathology of the Goethe University Hospital Frankfurt. HEK 293T and OCI-Ly3 cells were purchased from DSMZ (Braunschweig, Germany). For authentication, concordance of the VH gene sequences with published sequences was demonstrated by PCR analysis and sequencing. Cells were cultured in RPMI 1640 medium (Pan Biotech, Aidenbach, Germany), supplemented with 4 mmol glutamine and 10% FCS. Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density centrifugation (1,500 rpm for 30 min) and used on day 2 after preparation without stimulation. DH5α competent Escherichia coli (E. coli) were obtained from Thermo Scientific™ (Waltham, MA, USA) and used for general cloning and sub-cloning. TG1 E. coli were used for expression of the heavy-chain-only Fab-format neurabin-I BAR-body.

Monocytes were isolated using the Classical Monocyte Isolation Kit, human 130-117-337 from Miltenyi Biotec (Bergisch Gladbach, Germany) from PBMCs obtained from buffy coats of healthy, anonymous donors. Buffy coats were purchased from the Regional Center for Blood Donation and Blood Treatment, Łódź, Poland, as waste material. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany) and 10% human AB serum (PAN Biotech).

The human epithelial squamous cell carcinoma cell lines SCC-25 and A431 (both ATCC, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Pan Biotech, Germany) supplemented with 10% foetal bovine serum, 1% penicillin, 1% streptomycin and 1% glutamine (all Corning, Germany). Cells were kept under standard culture conditions at 37 °C, 95% humidity, and 5% CO₂ in a cell culture incubator (Binder, Germany). To establish an in vitro model of repeated gas plasma treatment, cells were exposed to gas plasma weekly in eight treatment cycles. Therefore, 1 × 10⁵ cells in 750 µl fully supplemented medium were seeded per well in a 24-well plate (Greiner Bio-One, Germany) and treated with gas plasma immediately. After 24 h of incubation, cells were harvested, transferred to a new cell culture flask, and cultivated until the treatment cycle of the following week. Cells and culture supernatants were collected for downstream analysis after single exposure and multiple treatment cycles for downstream analysis. Wild-type (WT) and repeatedly exposed (RE) cells after eight treatment cycles were cryopreserved for in vivo experiments. Due to the experimental complexity, as well as time intensive and elaborate study design, the long-term cell culture experiment was performed once for each cell line, representing one single biological replicate.

Before the CNS was taken, mice were intracardially perfused with 10 ml PBS. The CNS was minced with a razor blade, digested for 45 min at 37 °C in a shaking water bath (1 mg/ml Collagenase A, Roche; 0.1 mg/ml DNaseI, Merck Millipore; in RPMI-1640 medium, PAN Biotech) and subsequently homogenized through a 70 µm cell strainer. After washing with PBS, immune cells were isolated by percoll gradient centrifugation (30%/78% 1.13 g/ml, GE Healthcare) at 2500 rpm, 30 min, 4 °C, w/o brake, harvested from the interphase and washed twice with PBS.