M 2 mercaptoethanol

ES cells, EC cells and hybrids were harvested by treating the cells with 0.25% trypsin. The clumps of cells were transferred to a poly(2-hydroxyethyl methacrylate)-coated dish with DMEM/F12 supplemented with 20% KSR (Invitrogen), 2 mM l-glutamine, 1 × 10⁻⁴ nonessential amino acids, 1 × 10⁻⁴ M2-mercaptoethanol (Invitrogen) and 1% penicillin/streptomycin. The medium was changed every other day, and the EBs were harvested at days 3, 5, 7 and 9 to examine gene expression.

Study participants were tested for CD8⁺ T cell responses against CMV pp65 and immediate early-1 (IE-1) proteins using sets of overlapping peptides (Peptivator, Miltenyi Biotec, San Diego, CA, USA). Aliquots of 2 × 10⁶ PBMC in 1 mL lymphocyte medium (RPMI 1640 with 10% FCS, 100 IU/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, 10 mM HEPES buffer solution, and 2.0 × 10⁻⁵ M 2-mercaptoethanol from Invitrogen, Carlsbad, CA, USA) were stimulated with pooled CMV-pp65 (0.5 µg/mL) and IE-1 (0.5 µg/mL) peptide sets for 60 min at 37 °C (5% CO₂). Brefeldin A (Sigma-Aldrich) was then added to a final concentration of 10.0 µg/mL and the PBMC left for an additional 4 h before staining for surface markers and intracellular interferon-gamma (IFN-γ). Cells were washed twice in flow cytometry buffer consisting of PBS with 0.2% NaN₃, 5 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich), and 0.5% FCS, then stained with fluorescein isothiocyanate-conjugated anti-human CD3, clone BW264/56, and peridinin chlorophyll protein-conjugated anti-human CD8, clone BW135/80, (Miltenyi Biotec), for 20 min at 4 °C. Samples were kept in the dark, and after another wash with flow buffer, cells were fixed and permeabilized with InsideStain (Miltenyi Biotec) according to manufacturer’s instructions, then stained with allophycocyanin-conjugated anti-human IFN-γ, clone 4S.83 (eBioscience).