Fbs mem

As a growth medium, the Minimum Essential Medium Eagle (MEM, Sigma-Aldrich) commercial solution was enriched with 1 mM of penicillin, streptomycin, and 0.25 mM L-glutamine (Sigma-Aldrich). Two MEM-enriched solutions were used: one with 10% Fetal Bovine Serum (FBS-MEM) and one without (MEM, Sigma-Aldrich). Human epithelial lung cancer cell line (A549, CCL-185, passage number: 1) and fibroblast lung cell line (MRC-5, CCL-171, passage number: 30) were purchased from the American Type Culture Collection (ATCC). In FBS-MEM, cells were maintained as a monolayer culture at 37 °C in a humidified 5% CO₂ atmosphere. Cells were sub-cultured according to the protocols recommended to the cell’s phenotype and described previously in Jakubczak et al. [23 (link)]. Cells were detached from the culture flasks using 2 mL of 0.05% trypsin-EDTA solution (trypLE Express, Gibco) for 5 min, suspended in 6 mL of FBS-MEM, and centrifuged for 5 min at 1500 rpm (Universal 32 Tabletop Centrifuge, Hettich, Tuttlingen, Germany). The supernatant was removed, and cell pellets were resuspended in the proper amount of FBS-MEM to obtain the cell density of 1 × 10⁵ cells/mL.

Human blood was obtained from healthy (anonymous) donors at the Gulf Coast Regional Blood Center, Houston TX, and was purchased the Blood Center with an IRB exemption. The monocytes were obtained from buffy coat by gradient centrifugation using Ficoll-Paque (GE Healthcare Life Sciences). Non-adherent cells were removed and purified monocytes were incubated for 7 days in RPMI 1640 supplemented with 10% FBS and 50 ng/ml M-CSF to obtain macrophages (hereafter Human Primary Macrophages). Cells were washed with PBS twice and incubated overnight with 10% FBS-MEM supplemented with 1 μg/ml of LPS (Sigma, St. Louis, MO, USA). Cells were then washed with PBS twice and cultured with MEM-1% FBS for 48 h. The conditioned medium was harvested and filtered through a 0.22-μm filter to remove cell debris before being added to the CRC cell cultures.