L glutamine

Human peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll density gradient. It was performed using Ficoll-Paque Plus from Healthcare which was added to the blood in 1:2 dilutions. Around 1 × 10⁸ cells/mL were resuspended and rested during 2 or 3 h in X-VIVOTM medium from Lonza and 2% of human serum (Sigma Aldrich, Taufkirchen, Germany) (37 °C, 5%, CO₂, 70–80% humidity) in Corning^® cell culture 175 cm² flasks from Sarstedt. The isolation of lymphocytes was performed using a double-sided adhesion protocol. Non-adherent cells or lymphocytes were collected and cultured with completed RPMI 1640 medium (Biological Industries, Kibbutz Beit-Haemek, Israel) from 10% of inactivated foetal bovine serum (FBS), 1% of penicillin-streptomycin, and 1% of L-glutamine at 2 million of cells/mL (37 °C, 5% CO₂, 70–80% humidity) in 75 cm² flasks from Sarstedt (Nümbrecht, Germany).

First, 0.05% Trypsin-EDTA (1X) (Gibco, Sigma-Aldrich Company Ltd., Poole, UK), phosphate-buffered saline (PBS) solution tablets (prepared by dissolving one tablet in 200 mL of deionised water), Dulbecco’s Modified Eagle Medium (Gibco DMEM 1X, Sigma-Aldrich Company Ltd., Poole, UK), containing 4.5 g/L of glucose and 0.11 g/L of sodium pyruvate stored at 4 °C, antibiotics prepared with 100 µg/mL of streptomycin sulphate, 0.25 µg/mL of amphotericin B, and 100 U/mL penicillin G sodium, Gibco foetal bovine serum (FBS), thiazolyl blue tetrazolium bromide (MTT) powder, DMSO, menadione, DPPH (1,1-diphenyl-2-picrylhydrazyl), methanol, fluorescein diacetate (FD), propidium iodide (PI), and ascorbic acid were all purchased from Sigma-Aldrich (Poole, UK). L-glutamine, Gibco trypan blue stain (0.4%), 96-well microplates, 24-well microplates, and fluorescence-activated cell sorting (FACS) tubes were purchased from Sarstedt (Sarstedt Ltd., Nümbrecht, Germany). A ROS-Glo™ H2O2 Assay Kit was purchased from Promega (Promega UK Ltd., Southampton, UK). The antioxidant scavenging activity was measured by the use of a microtiter plate reader (Infinite 200 Pro, Tecan Trading, Männedorf, Switzerland). All other materials were obtained from Thermo Fisher Scientific (Loughborough, UK) and used as received.

Colon cancer cells (Caco‐2, HT‐29) and fibroblasts (CRL2072) were used to study the biological activity of the extracts. Cells were maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% heat‐inactivated fetal bovine serum, 2 mM l‐glutamine, and penicillin (100 U/ml)/streptomycin (100 g/ml) in flasks (75 cm² surface area; Sarstedt, Spain) at 37°C under 40% humidity with a 5% CO₂ atmosphere.