Rpmi 1640 with l glutamine

The following tumor cell lines were used: the PC-3 cell line (ATCC Number: CRL-1435^TM) was kindly provided by Dr. Cyrill Bussy (Centre for Drug Delivery Research, UCL School of Pharmacy, United Kingdom), the LNCaP clone FGC cell line (ATCC Number: CRL-1740^TM) was purchased from Sigma–Aldrich, United Kingdom, and was obtained from the American Type Culture Collection (ATCC). Human Dermal Fibroblasts, adult (HDFa) catalog number C-013-5C was kindly provided by Dr. George Pasparakis (Department of Pharmaceutics, UCL School of Pharmacy). Antibiotic-antimycotic, penicillin-streptomycin, trypsin-EDTA (0.25% trpysin, EDTA 4Na), phosphate-buffered salines (PBSs), fetal bovine serum (FBS), and Eagle’s Minimum Essential Medium (EMEM) were purchased from GIBCO^®, United Kingdom (UK). RPMI 1640 with L-glutamine was purchased from Lonza, United Kingdom. All other chemicals were obtained from Sigma–Aldrich^®, United Kingdom, unless stated otherwise.

The THP-1 cell line was kindly provided by Dr. Nuno Santos (CBME, University of Algarve, Faro) and cells were cultured according to ATCC instructions in the RPMI Growth Medium (RPMI 1640 with L-Glutamine (Lonza, Basel, Switzerland), 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Waltham, MA, USA) and 1% Pen-Strep (P/S, Gibco, Waltham, MA, USA). Differentiation into macrophagic THP-1 (THP-1 MoM) was achieved by culturing cells in 25 ng/mL PMA (Sigma Aldrich, Burlington, MA, USA) in complete RPMI for 48 h.
Human aortic VSMCs (VSMC) were derived from tissue explants as described previously [45 (link)], and used between passages 4 and 12. VSMCs were maintained in M199 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) of P/S.
The primary human articular chondrocytes were commercially acquired (Lonza, Visp, Switzerland), and cultured in advanced Dulbecco’s modified eagle’s medium (Adv DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) of heat-inactivated FBS, 1 mM of L-Glutamine (L-Gln, Invitrogen, Waltham, MA, USA) and 1% (v/v) of P/S.
All cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂, and experiments were performed on confluent VSMC and chondrocyte cells, using an average of 1 × 10⁶ cells/mL of THP-1 MoM.

Human NSCLC cell lines (A549, H1299, H157, H358, H460 and H520) were obtained from the American Type Culture Collection and the European Collection of Cell Cultures. All the cell lines were grown in RPMI complete medium: RPMI-1640 with L-glutamine (Lonza) supplemented with 10% Fetalclone III (Hyclone) and 1% Penicillin-Streptomycin (Lonza). Cells were authenticated by PCR analysis on the basis of their known mutations (COSMIC database).
Immortalized human bronchial epithelial cells (HBEC-3KT) were a kind gift from Dr. John D. Minna (University of Texas Southwestern Medical Center, Dallas, Texas). Cells were derived from a donor after obtaining informed consent on institutional review board-approved protocols. These non-tumorigenic cells were obtained from bronchial cells by overexpressing CDK4 and TERT [15 (link),16 (link)]. They were grown in serum-free Keratinocyte-SFM (Gibco) supplemented with bovine pituitary extract (Gibco), recombinant EGF (Gibco) and 1% Penicillin-Streptomycin. Cells were cultured in a humidified incubator at 37°C and 5% CO₂.

C10 media was used for all cell culture experiments. C10 media consisted of RPMI 1640 with L-glutamine (Lonza, Basel, Switzerland) containing 10% FCS (non-Hi FCS; Highveld Biological, JHB, SA), 2% L-glutamine, 1% HEPES, 1% NaPy, 1% NEAA (all from Lonza, Basel, Switzerland). Media was sterile filtered through the Filtermax 500 ml (Techno Plastic Products, Trasadingen, Switzerland). IL-2 (PeproTech, Rocky Hill, NJ, USA), was added to C10 media prior to use at a final concentration of 0.01 μg/ml.

Naïve splenic CD8⁺ T cells were magnetically enriched using an AutoMACS pro (Miltenyi Biotec) using the CD8a T cell isolation kit supplemented with anti-CD44-biotin (eBioscience). The cells were cultured at 2 × 10⁵ cells/ml in RPMI-1640 with L-glutamine (Lonza, Basel, Switzerland) + 10% FCS 1:1 with α-CD3/α-CD28 immobilized on beads (Miltenyi), 10 U/ml rmIL-2 (eBioscience). The purity of isolated naïve T cells was assessed by flow cytometry and was >95% in all experiments. Lymph node (LN) stromal cells were obtained by dissociating lymph nodes in Liberase TL enzyme mixture (Roche, Basel, Switzerland) using GentleMACS tissue dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). When lymph nodes were fully digested lymph node stromal cells were negatively enriched using anti CD45 and anti Ter119 magnetic beads (Miltenyi Biotec). Stromal cells were passaged 5–10 times in alpha-modified MEM (Sigmaaldrich, St.Louis, Mo) with 20% FCS. Naïve T cells were cultured with lymph node stromal cells in 1:10 ratio in 96 well-round plates.