Flat bottomed plates

According to the previous reports^{11 (link),16 (link)}, DCs (10⁵) were cultured with or without IMQ (10 μg/ml; Invivogen) for 5 hrs in 96-well flat-bottomed plates (BD Bioscience).

CD45.1⁺OT-I CD8⁺ T cells (10⁵) or CD45.1⁺OT-II CD4⁺ T cells (10⁵) were cultured with the irradiated (15 Gy) CD8α⁻ cDCs (1.25 × 10³–10⁴) in the presence of OVA_257–264 peptide (1 pM–1 nM) or OVA_323–339 peptide (ISQAVHAAHAEINEAGR; 1 nM–1 μM) for 3 days in 96-well flat-bottomed plates. Alternatively, CD4⁺ T cells (2 × 10⁵) were cultured with irradiated (15 Gy) CD11c⁺ DCs (2 × 10⁴) in the presence or absence of OVA protein (1 mg ml⁻¹, Sigma-Aldrich) or MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; 1 μM) for 3 days in 96-well flat-bottomed plates (BD Biosciences). [³H]thymidine (GE Healthcare) incorporation was measured on day 3 for the last 18 h. In another experiment, the cells and the culture supernatants were collected to detect the production of cytokines.

CD45.1⁺ OT-I CD8⁺ T cells (10⁵) or CD45.1⁺ OT-II CD4⁺ T cells (10⁵) were cultured with the irradiated (15 Gy) cDCs or pDCs (10⁴) in the presence of OVA_257–264 peptide (1 nM) or OVA_323-339 peptide (ISQAVHAAHAEINEAGR; 1 μM) for 3 days in 96-well flat-bottomed plates. Alternatively, CD4⁺ T cells (2×10⁵) were cultured with irradiated (15 Gy) CD11c⁺ DCs (2×10⁴) in the presence or absence of OVA protein (1 mg/ml, Sigma-Aldrich) for 3 days in 96-well flat-bottomed plates (BD Biosciences). [³H]thymidine (GE Healthcare) incorporation was measured on day 3 for the last 18 hrs. In another experiment, the cells and the culture supernatants were collected to detect the production of cytokines.