Heat inactivated fetal calf serum fcs

MSCs from human dental pulp were expanded as described previously [8 (link)] and transduced with lentiviral vectors pWP-green fluorescent protein (GFP-MSCs) or pWP-HIF-GFP (HIF-MSCs) as previously described [7 (link)]. Transduction efficiency was evaluated by flow cytometry (Coulter EPICS XL flow cytometer; Beckman Coulter) to determine the percentage of GFP-positive cells. The percentages of infection obtained were normally around 90%. Human dental pulp MSCs (n = 3; Inbiobank) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Sigma-Aldrich, Spain) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco, Life Technologies, Thermo Fisher Scientific). Cells were grown until confluence and subsequently subcultured up to 12 passages. For stimulation assays, 5 × 10⁴ MSCs were seeded in six-well flat-bottom culture plates and, after 3 days, media were replenished and supplemented or not with 10 ng/ml recombinant human interferon (rhIFN)-gamma (Invitrogen). After 15 h, cells were lysed for subsequent analysis by quantitative polymerase chain reaction (qPCR).

The human NB cell lines SK-N-AS, SK-N-SH, and SK-N-BE(2)-C were purchased from ATCC (LGC Standard, Wesel, Germany). GI-M-EN, SH-SY5Y, IMR32, LAN5, and KELLY cells were from DSMZ (Braunschweig, Germany), and NB69 cells were from ECACC (Salisbury, UK). IMR5 and NLF cells were a gift from G. M. Brodeur (CHOP, Philadelphia, PA, USA). SK-N-AS and SK-N-SH cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Paisley, UK) supplemented with 10% of heat-inactivated fetal calf serum (FCS; Gibco), SH-SY5Y cells in DMEM with 20% FCS, and SK-N-BE(2)-C cells in a 1:1 mixture of EMEM and Ham’s F12 (Gibco) with 10% FCS. GI-M-EN, SH-EP, NLF, and KELLY cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) with 10% FCS, NB69 in RPMI 1640 medium with 15% FCS, and IMR5, IMR32, and LAN5 cells in RPMI 1640 medium with 20% FCS. All media were supplemented with 2 mM of l-glutamine (Gibco) and 100 U/ml of penicillin/streptomycin (Gibco). The authenticity of cells was verified by short tandem repeat (STR) profiling using the Cell Line Authentication Kit (Sigma-Aldrich, Taufkirchen, Germany). Cells were tested for mycoplasma contamination using the LookOut Mycoplasma PCR Detection Kit (MP0035, Sigma-Aldrich).

Venous peripheral blood was obtained from healthy donors with known HLA types. Each blood donor had signed a written consent form, allowing for the use of his or her blood for research purposes. The procedure was approved by local ethics legislation. The blood was collected in heparinized tubes (Vacuette, Lithium Heparin, Greiner Bio One, Frickenhausen, Germany). Isolation of peripheral blood mononuclear cells (PBMCs) was accomplished by using Lymphoprep ™ gradient centrifugation (Axis-Shield, Oslo, Norway). The PBMCs were either used directly after the isolation or stored at −140 °C in a storage medium (RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA), 40% heat-inactivated fetal calf serum (FCS) (Gibco), 10% dimethyl sulfoxide (Merck Millipore, Darmstadt, Germany), 100 U/mL penicillin/10 µg/mL streptomycin (Ampliqon, Odense, Denmark)). The CD4+ T cells were isolated from PBMCs using either a Dynabeads^® CD4 Positive Selection kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) or a Dynabeads^® Regulatory CD4+ CD25+ T Cell Kit (Invitrogen), using only the CD4+ isolation part. The entire procedure was carried out according to the manufacturer’s guidelines. The purity of the isolated cells was evaluated by flow cytometry.