Purified DCs (1 × 104 in 200 μL culture medium) were stimulated with different TLR ligands at the concentration mentioned above.
DCs were enriched by lineage (CD3, CD14, CD16, CD19) depletion, then lineage negative cells (2 × 105 in 200 μL culture medium) were stimulated with TLR ligands or viruses for 4 h followed by another 2 h in the presence of Golgi Blocker (BD Biosciences, Franklin Lakes, NJ, USA).
Differentiated T37i cells, starved for 18 h in minimum medium, were stimulated with 100 ng/ml PRL, during 0, 5 and 15 min; cells were washed twice with ice-cold PBS and lysed as previously described [18].
PBMC (6×106 cells/ml) in culture medium were stimulated for 18 to 20 hours with peptides (PfCS102, 10 µg/ml, plus HLA-A2 and/or HLA-A3 CTL peptides, 5 µg/ml each) in the presence of brefeldin A (10 µg/ml, Sigma) during the last four hours of culture.
D407 cells, starved for 24 hours in serum-free medium, were stimulated with 100 nM Tat for different time durations.
Jurkat E6.1 cells (2 × 10 per ml RPMI 1640 medium) were stimulated with gal-1 (20 μg/ml) at 37°C and washed once with ice-cold phosphate-buffered saline (PBS).
Inducible TIG1A, TIG1B, and control stable HCT116 cell clones plated overnight in 10-cm dishes in growth medium were stimulated with growth medium containing 5 nM MFP for 24 h.
Cells grown in serum-free medium were stimulated by TNFα (50 ng/ml) or epidermal growth factor (EGF; 100 ng/ml) for time points indicated in the relevant figures.
Aliquots of 1 × 10 cells per mL of medium were stimulated with LPS (0.01 1000 ng/mL) for 16 h in HydroCell Surface 24-well plates (1 mL/well) in humidified atmosphere (5 vol% CO2, 37°C).
