Murine llc cells

Mouse L929 fibroblasts, 4T1 murine mammary carcinomas, and murine LLC cells were obtained from ATCC (Manassas, VA, USA). E0771 murine mammary carcinoma cells[34 (link)], ID8 ovarian carcinoma cells[35 (link)], and HC11 mouse mammary epithelial cells [36 (link)] were kind gifts from Drs. David Waisman, Craig McCormick, and Hyo Sung Ro (all from Dalhousie University, NS, Canada) respectively. All cell lines were grown in T75 tissue culture flasks (Sarstedt, QC, Canada) and maintained at 37°C and 5% CO₂ in DMEM containing 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 5 mM HEPES (ph 7.4), hereafter referred to as “DMEM” unless otherwise indicated. At 90% confluence, cells were passaged by detachment with trypsin-EDTA, followed by centrifugation at 500 x g for 5 and then resuspension in fresh DMEM. Freshly isolated BMDMs were cultured at 37°C at 5% CO₂ in RPMI-1640 medium containing 10% FCS (v/v), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 5 mM HEPES (ph 7.4), hereafter referred to as “RPMI-1640” unless otherwise indicated. Prior to experimental use, BMDMs were detached with EDTA (10 mM in PBS), centrifuged at 500 x g for 5 min, and resuspended in fresh RPMI-1640 before cell seeding.

Human CD14⁺ monocytes were purified from one-day-old buffy coats, derived from healthy donors (Belgian Red Cross, Mechelen, Belgium), through density gradient centrifugation and positive selection (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described [25 (link)]. Human neutrophils were isolated from fresh blood derived from healthy donors via density gradient centrifugation as previously described [26 (link)].
The murine macrophage cell line RAW264.7 [American Type Culture Collection (ATCC), Manassas, VA, USA] was grown in Dulbecco's modified Eagle's medium (DMEM; Lonza, Verviers, Belgium) supplemented with glucose (4.5 g/l), 1 mM sodium pyruvate and 10% fetal calf serum (FCS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The murine fibroblast cell line L929 (ATCC) was grown in minimum essential medium (MEM) Rega (Thermo Fisher Scientific) supplemented with 10% FCS. Murine LLC cells (ATCC) were grown in DMEM supplemented with glucose (4.5 g/l) and 10% FCS. Murine peritoneal cells were extracted from healthy female NMRI mice (Charles River, Wilmington, MA, USA) that were kept in a specific pathogen-free environment. After euthanizing the mice, peritoneal lavages were carried out with 5 ml of phosphate-buffered saline (PBS) supplemented with 20 U/ml of heparin (LEO Pharma, Lier, Belgium) and 2% FCS.