Rpmi 1640

MPs were first stained with an anti-CD27- BV510 antibody (BD Biosciences) at 4°C for 30 minutes and then washed by centrifugation for 1 hour at 100,000 x g and 4°C, to eliminate the antibodies that had not bound to MPs. MPs were resuspended in filter-sterilized medium (passage through a filter with 0.1 μm pores) and the CD27⁺ MPs present in the MP preparation were counted.
PBMCs were isolated by Ficoll density gradient centrifugation. We cultured 2 x 10⁴ PBMCs for 18 hours with quantified CD27-expressing MPs. We added various amounts of the bulk MP preparation to the culture medium (filter-sterilized, passage through a filter with 0.1 µm pores) such that we obtained ratios of PBMCs to CD27-expressing MPs of 1:1, 1:10 and 1:20 (PBMC: CD27-expressing MPs).
The culture medium consisted of RPMI 1640 supplemented with 5% FBS (Dutscher, Bernolsheim, France), 2 mM L-glutamine, 100 µg/ml penicillin/streptomycin, MEM non-essential amino acids solution (1X), and 1 mM sodium pyruvate (all from Life Technologies, Carlsbad, CA).
Following coculture, the PBMCs were harvested for flow cytometry to assess their co-expression of CD27 from MPs (BV510-labeled). PBMCs were stained with anti-CD4-APC-H7 and anti-CD3-PE (BD Biosciences) antibodies. After staining (4°C, 30 minutes), the cells were washed twice with PBS.

MDA-MB-231 (human mammary adenocarcinoma), A549 (human lung adenocarcinoma), SK-HEP-1 (human hepatocellular carcinoma), DU145 (human prostate adenocarcinoma) and the two human colon adenocarcinomas HCT116 and SW480 cell lines were cultured with high-glucose Dulbecco's modified Eagle's medium provided by Dutscher (Dutscher, Brumath, France) supplemented with 10% fetal bovine serum (Dutscher). PANC-1 (human pancreatic carcinoma) cell line was cultured in RPMI 1640 (Roswell Park Memorial Institute medium) provided by Dutscher and supplemented with 10% fetal bovine serum. All these cell lines were grown in an incubator at 37 °C and 5% CO₂.

MSC-2 cell line is CD11b⁺/Gr-1⁺ immortalized MDSC obtained from CT-26 tumor-bearing BALB/c mice^{21 (link)}.
EL4 thymoma cells and MSC-2 cells were cultured at 37 °C under 5% CO₂ in RPMI 1640 with 10% (v/v) heat inactivated fetal bovine serum penicillin, streptomycin, amphotericin B antibiotic cocktail, all from Dutscher (Dutscher, Brumath, France). Cell lines were authenticated by examination of morphology and growth characteristics and confirmed to be mycoplasma free. MSC-2 cells were treated with FAs (20–60 µM) bound to FA free-bovine serum albumin (ratio 4:1) (BSA, Sigma-Aldrich) 3 h prior to 5-FU treatment at 1 µM or lipopolysaccharide (LPS) (O55:B5, Sigma-Aldrich) at 100 ng/ml.

CT26 American Type Culture Collection (ATCC) murine colon cancer cells (USA) were cultured in RPMI 1640 (Dutscher, France) + 10% fetal calf serum (Dutscher, France) (37 °C, 5% carbon dioxide and 95% humidity). B16-F10 murine melanoma cancer cells (USA) were cultured in DMEM (Dutscher, France) + L-Glutamine + red phenol + glucose (4.5 g/l) + 10% fetal calf serum (Dutscher, France) (37 °C, 5% carbon dioxide and 95% humidity).
The day before mice were injected with cancer cells. These cells were contacted with trypsin and diluted to ½. The unit injection included 5 × 10⁵ CT26 cells in 100 μl of NaCl, or 1 × 10⁶ B16-F10 cells 100 μl of NaCl, in performed subcutaneously on the right flank of immunocompetent BALB/c female and C57BL female mice and 8-week immunosuppressed athymic BALB/c nude mice (Charles River Laboratories, Saint-Germain-des-Monts, France). During the entire duration of the experiment, mice were housed in our approved animal facility (Centre Georges-François Leclerc, Dijon, FRANCE). The mice were sacrificed by cervical dislocation after Isoflurane 2.5% anesthesia as soon as a limit point was reached (Tumoral Volume (TV) ≥1500 mm3, pain, significant necrosis).
Before experimentation, the small animal ethics committee and the Ministry of Higher Education and Research validated the project.