Immature DCs are allowed to adhere onto fibronectin and subsequently incubated with FITC-labeled yeast or spores for 5, 15, 30 minutes at 37°C. At the end of the incubation period, the samples are fixed in 4% PFA, permeabilized in Methanol, and labeled for DC-SIGN and/or MR using specific mAb and isotype-specific fluorescent secondary Abs. Samples are analyzed using a Zeiss LSM 510 confocal microscope. Alexa647-conjugated goat-anti-mouse IgG, and Alexa568 goat-anti-mouse IgG2b from Molecular Probes.

All the individual microcultures are then screened for recognition of autologous EBV-B cells transduced with a retrovirus encoding the protein. Non transduced EBV-B cells are used as control stimulator cells.

Activated T cells are detected by their expression of CD137.

The DC populations were CD123+, CD11c+CD16+, CD11c+BDCA1+, CD11c+BDCA3+ DC.