24 well flat bottomed tissue culture plates

T cells were purified from the LN and spleen of mice. Cells were purified after a depletion step using antibodies against CD11b (M1/70), F4/80, Ter-119, Gr-1 (RB6), MHC class II (M5/114), and CD4 (GK 1.5) or CD8 (YTS 169.4), followed by incubation with anti-rat IgG-coupled magnetic beads (Dynal Biotech) following the manufacturer’s protocols. T cell preparations were 90–95% pure as determined by flow cytometry. Purified naïve CD4⁺ or CD8⁺ T cells were plated at 10⁶ cells per well in 2-ml 24-well flat-bottomed tissue culture plates (Falcon) coated with anti-CD3(145-2C11) and anti-CD28 (37.51). Alternatively, OT-I cells were plated at 10⁶ cells per well in 2-ml 24-well flat-bottomed tissue culture plates (Falcon) with 1 μg/ml SIINFEKL peptide.

After purification, 0.5 × 10⁶ cells/mL were cultured into either 24-well flat-bottomed tissue culture plates (Falcon Inc., USA) or round-bottomed polypropylene tubes (Falcon Inc., USA), in RPMI-1640 culture medium supplemented with 10% HS. For differentiation of monocytes into moDCs, the culture medium was supplemented with 300 or 500 UI/mL of hrGM-CSF and 300 or 500 UI/mL hrIL-4. Both media replacements and control (baseline) cultures were set up as described earlier. In tissue culture plates, cells were detached and collected using a 5 mL syringe rubber plunger in gentle, one-way movements, followed by washing the wells with ice-cold PBS pH 7.4 and gently transferring cells into round-bottomed polypropylene tubes (Falcon Inc., USA) for staining procedure. Samples were stained with the following mAbs: anti-hCD14-PE/Dazzle 594 (Dilution 1:100; Clone HCD14; BioLegend, USA; cat 325634) and anti-hCD209-PE (Dilution 1:200; Clone 9E9A8; BioLegend, USA; cat 330106). The precise staining procedure and flow cytometer analysis performed are described in the following section.