E coli 055 b5

Rat enteroglial cell line CRL-2690 (EGCs) and mouse macrophage cell line RAW 264.7 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)–high glucose (containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin) and maintained at 37 °C in a humidified incubator of 5% CO₂.
EGC were seeded in 6-well plates and cultured in the medium containing 0 or 1 nmol/L RA (Sigma-Aldrich, Burlington, MA, USA) and 0 or 1 μg/mL LPS (E. coli 055: B5, Sigma-Aldrich). After 24 h incubation, the supernatants were collected to quantify IL-1β production. The adherent EGCs were harvested for total RNA extraction and total protein extraction.
Rat RAW 264.7 cells were cultured in 6-well plates with medium containing 0 or 1 nmol/L RA (Sigma-Aldrich), 0 or 1 ng/mL GDNF (Sigma-Aldrich), and 0 or 1 μg/mL LPS (E. coli 055: B5, Sigma-Aldrich). After 16 h culture, the supernatants were collected to measure IL-1β and COX-2 production. The adherent macrophages were harvested for total RNA extraction.

Since the non-toxicity of the extract is a fundamental pre-requisite, we first tested the cytotoxicity of the CSE in a concentration range of 0.0002–2 mg / ml. No toxicity was showed at any doses tested.
pAECs were seeded in a 96 wells plate (approximately 3 × 10³ cells/well) and exposed to increasing doses of Cucumis sativus L. (CSE) (0.02; 0.2; 2 mg/ml) in presence of lipopolysaccharide (LPS) (10 μg/ml) (E. coli 055:B5, Sigma-Aldrich Co, St Louis, MO, USA) for 24 h. Cytotoxicity was evaluated by trypan Blue exclusion dye using Countess® II FL Automated Cell Counter (Life Technologies).

Purified T cells were labeled with CFSE (Molecular probes, Life Technologies, Carlsbad, CA, USA) and cultured in 96-well flat bottom plates (Nunclon Delta Surface, Thermo Scientific, Waltham, MA, USA) for 5 days—or otherwise if indicated—at 37°C in IMDM medium (Gibco, Life Technologies, Carlsbad, CA, USA), supplemented with 10% (v/v) fetal calf serum (Bodinco, Alkmaar, The Netherlands), 10⁴ U/mL penicillin, 10 ng/mL streptomycin, 200 mM glutamine, and 0.035% (v/v) β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, USA). Proliferation was induced by anti-CD3 (clone 1XE [IgE isotype] hybridoma supernatant, 1:1,000, Sanquin, Amsterdam, The Netherlands) and anti-CD28 (clone 15E8 [IgG1 isotype] at 5 μg/mL, Sanquin) monoclonal antibodies (moAbs; at 20,000 T cells/well). Mature neutrophils from blood or neutrophil progenitors from bone marrow were added in a 1:3 ratio (60,000 neutrophils/well), in the presence or absence of neutrophil-activating stimuli: fMLF (1 μM, Sigma-Aldrich), TNFα (10 ng/mL, Peprotech EC, London, UK) or LPS (20 ng/mL, E. coli 055:B5, Sigma).
After 4–6 days, T cell proliferation, indicated by CFSE dilution, was analyzed by flow cytometry. Cells were harvested from the culture plates and stained with APC-labeled anti-CD4 (clone SK3, BD Biosciences, San Jose, CA, USA) and PerCPCy5.5-labeled anti-CD8 (clone SK1, Biolegend, San Diego, CA, USA) antibodies.