The B:T conjugation assay (26 (link), 79 (link)) was performed as follows. Empty-GFP (CD45.11) and Mef2d-GFP (CD45.12) OTII CD4 T cells were prepared. Splenic B cells, isolated from C57BL/6J mice, were stimulated with LPS (1 μg/mL) for two days. Activated B cells were labeled with 50 μM CMF2HC (Invitrogen) for 30 minutes at room temperature, followed by a brief pulse with OVA323–339 peptide (0, 1, or 10 μg/mL) for 30 minutes at 37 °C. 1.25 × 105 empty-GFP and 1.25 × 105 Mef2d-GFP OTII CD4 T cells were co-cultured with 5.0 × 105 B cells for 20 or 30 minutes at 37 °C.
The DC:T conjugation assay was done in similar manners to the B:T conjugation assay with following modifications. Splenic DCs, after isolation with CD11c microbeads (Miltenyi Biotec) and subsequent staining with CD11c antibody for 20 minutes at 4 °C, were pulsed with OVA323–339 peptide for 2 hours at 37 °C. 2.5 × 105 DCs were co-cultured with empty-GFP and Mef2d-mAmetrine OTII CD4 T cells.
Conjugate formation of the respective OTII CD4 T cells with B cells or DCs was analyzed based on congenic molecule (CD45.11 vs. CD45.12) expression or based on GFP and mAmetrine expression among CD4 T cells in the conjugates.
The DC:T conjugation assay was done in similar manners to the B:T conjugation assay with following modifications. Splenic DCs, after isolation with CD11c microbeads (Miltenyi Biotec) and subsequent staining with CD11c antibody for 20 minutes at 4 °C, were pulsed with OVA323–339 peptide for 2 hours at 37 °C. 2.5 × 105 DCs were co-cultured with empty-GFP and Mef2d-mAmetrine OTII CD4 T cells.
Conjugate formation of the respective OTII CD4 T cells with B cells or DCs was analyzed based on congenic molecule (CD45.11 vs. CD45.12) expression or based on GFP and mAmetrine expression among CD4 T cells in the conjugates.
