Rpmi 1640

All tested monocarbonyl analogs of CUR were dissolved in a cell culture grade DMSO (Sigma Aldrich, Poznan, Poland) followed by ultrasonic mixing for 3 min at rt before use. Immediately after sonication, the appropriate amount of each compound was transferred to a medium to acquire a working concentration of 3000 μg/mL and mixed. The compounds were diluted in X-VIVO 10 (without Phenol red and Gentamycin, Lonza, Koln, Germany) with supplemented with a 10% fetal bovine serum (FBS, PanBiotech) for a 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, an RPMI 1640 (PanBiotech, Aidenbach, Germany) for an endotoxin chromogenic assay, or an RPMI1640 supplemented with 10% FBS for a cytokine assay. Working concentrations of all analyzed compounds were prepared using twofold serial dilutions in an appropriate medium to obtain series of 11 dilutions (from 1.46 to 1500 − μg/mL).

291PC,13 (link) A20 (ATCC, TIB-208), Phoenix-A (ATCC, CRL-3213), and EBV-LCL were cultured in RPMI 1640 (PAN-Biotech) with 10% FCS, 40 IU/mL penicillin, 40 µg/mL streptomycin, 2 mM l-glutamine, 0.4% vitamin solution, 50 µM β-mercaptoethanol, 1% minimal essential media, and 1 mM sodium pyruvate (all Gibco, Thermo Fisher Scientific). In murine splenocyte culture 50 IU/mL recombinant interleukin (IL)-2 (Proleukin) were added. For in vitro expansion of Marilyn-derived CD4+ splenocytes, cells were activated in anti-CD3 (3 µg/mL; 145–2 C11; Biolegend) and anti-CD28 (2 µg/mL; 37.51; Biolegend) coated plates in the presence of 5 IU/mL IL-2 for 24 hours, harvested and subsequently expanded with addition of 10 IU/mL IL-15 (Biolegend) for 7 days.14 (link) To generate immature dendritic cells monocytes were isolated by adherence and cultured for 5 days in the presence of IL-4 (500 IU/mL; Peprotech), GM-CSF (100 IU/mL; Miltenyi) and TGF-beta (5 ng/mL; Peprotech).
Human T-cells were cultured in RPMI 1640, as mentioned, but with 5% human serum (PAN-Biotech), 5% FCS, and 100 IU/mL IL-2 (Proleukin). Every 11–14 days, T cells were restimulated with 50 Gy irradiated allogeneic feeder cells and 0.8 µg/mL phytohemagglutinin (PHA) (Oxoid), as previously described.15 (link) Culture supernatants of cell lines were regularly tested for mycoplasma contamination (Minerva Biolabs) by PCR.

Human breast epithelial adenocarcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-436), and a human ovarian adenocarcinoma cell line (OVCAR-5) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human breast ductal carcinoma cell line; HCC1937 and HCC1937 reconstituted with wild type BRCA1 (HCC1937/wt BRCA1) were kind gifts from Dr. Grant Mc Arthur, Peter MacCallum Cancer Centre, VIC, Australia. All the cell lines were maintained in culture medium containing 10 % Fetal Bovine Serum (FBS) (PAN-Biotech GmBH, Aidenbach, Germany), 0.1 g/L Streptomycin (Sigma Aldrich, St. Louis, MO, USA) and 100U/L Penicillin in a humidified incubator at 37 °C in 5 % (v/v) CO₂. MCF7, MDA-MB-231 and OVCAR-5 were maintained in Dulbecco’s Modified Eagles’ Medium (Gibco, Carlsbad, CA, USA), MDA-MB-436 in RPMI 1640 (PAN-Biotech GmBH, Aidenbach, Germany) and HCC1937 and HCC1937/wt BRCA1 in RPMI 1640 with insulin (5 μg/ml). Passage numbers post thawing of the cell lines were noted and experiments were carried out within a maximal passage number (<30).