2 mercaptoethanol

Splenocytes were stimulated in vitro with 50 ng/ml PMA and 1 μg/ml ionomycin (both Sigma-Aldrich) for 4 h at 37°C in the presence of both Brefeldin A (GolgiPlug) and Monensin (GolgiStop, both BD Biosciences). As GolgiPlug and GolgiStop were used together, half the amount recommended by the manufacturer where used, as suggested previously [44 ]. Cells were stimulated in complete^mouse RPMI medium (RPMI 1640 supplemented with 10% (v/v) FCS (Corning), 1% (v/v) Pen-Strep-Glutamine (10.000 U/ml penicillin, 10.000 μg/ml streptomycin, 29.2 mg/ml L-glutamine (Gibco)) and 50 μM 2-mercaptoethanol (AppliChem), containing 0.42 mM Ca²⁺). For human studies, freshly isolated PBMCs or expanded cell populations were stimulated in vitro with 25 ng/ml PMA and 1 μg/ml PMA for 4 h at 37°C in the presence of Brefeldin A or monensin. Cells were stimulated in complete^human RPMI medium (RPMI 1640 supplemented with 10% (v/v) FCS, 1% (v/v) Penicillin/streptomycin, 1 mM sodium pyruvate (Lonza), 1% (v/v) non-essential amino acids (Cegrogen), 15 mM HEPES buffer (Sigma-Aldrich), and 55 μM 2-mercaptoethanol (AppliChem). Considering the Ca²⁺ found in the FCS (3.9 mM) [21 (link)] the supplemented RPMI medium contained approx. 0.8 mM Ca²⁺ (RPMI^norm). To obtain the RPMI^suppl medium containing elevated Ca²⁺ concentrations, 1 mM CaCl₂ (Sigma-Aldrich) was added to the RPMI^norm medium (i.e. approx. 1.8 mM Ca²⁺).

Human Vδ2⁺ T cells were expanded from PBMCs of healthy donors similar to published protocols [41 (link)–43 ]. Briefly, freshly isolated PBMCs (1 x 10⁶ cell/ml, 5 ml/well) were cultured with 5 μM Zoledronic acid (Zometa, Novartis) in the presence of 100 IU/ml human recombinant IL-2 (Proleukin, Novartis) for 13 days. IL-2 was replenished every other day and from day 6 onwards the concentration was increased to 200 IU/ml. The cultures were performed in RPMI1640 (Gibco or Lonza) supplemented with 5% (v/v) Human AB serum (Sigma-Aldrich), 1% (v/v) Penicillin/Streptomycin (Gibco), 1 mM sodium pyruvate (Lonza), 1% (v/v) non-essential amino acids (Cegrogen), 15 mM HEPES buffer (Sigma-Aldrich), and 55 μM 2-mercaptoethanol (AppliChem).

Human iNKT cells were expanded from resting PBMCs of healthy donors as described before [41 (link)]. Briefly, freshly isolated PBMCs (1 x 10⁶ cell/ml, 5 ml/well) were treated with 100 ng/ml αGalCer (KRN7000, Avanti Polar Lipids) and cultured for 13 days. 20 IU/ml human recombinant IL-2 (Proleukin, Novartis, Basel, Switzerland) was added to the cultures every other day starting from day 2. From day 6 onwards, the concentration of IL-2 was increased to 40 IU/ml. At the end of the expansion, an aliquot of each sample was collected and analysed for iNKT cell expansion by flow cytometry. Expansion was done in RPMI 1640 (Gibco, Waltham, MA, USA, or Lonza, Basel, Switzerland) supplemented with 5% (v/v) Human AB serum (Sigma-Aldrich, St. Louis, MO, USA), 1% (v/v) Penicillin/Streptomycin, 1 mM sodium pyruvate (Lonza), 1% (v/v) non-essential amino acids (Cegrogen Biotech, Stadtallendorf, Germany), 15 mM HEPES buffer (Sigma-Aldrich), and 55 μM 2-mercaptoethanol (AppliChem, Darmstadt, Germany).