The yeast oligonucleotide array is constructed using the S. cerevisiae Genome Oligo Set ™ (Operon Technologies, CA, USA) composed of 6240 optimized oligonucleotides (70mers) each representing one yeast gene.

Monocytes are isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch- Gladbach,Germany).

The cells are collected and the pellets are resuspended in 3.6 ml AE Buffer (AE=50mM NaAcetate pH 5.2, 10mM EDTA) and then transferred in 15 ml tubes wit 200 µl 10% SDS (w/v). 2 ml of preheated acid phenol (pH 4.3) are added and the falcon is mixed by vortex.

Serial diluition of yeast cells and preparations are added to the MoDCs.

Monocyte-derived DCs are added at a final concentration of 5x105 cells/ml into 96-well plates.

The samples are centrifugated at 5000 rpm in an tabletop centrifuge.

PBMC are isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM).

Different strains of Saccharomyces cerevisiae are cultured and collected in different conditions: different growth phase (exponential and stationary phase) different growth media (standard and promoting pseudohyphal growth) different cell form (spheroplast, spore and whole cell)

Cells are cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).

Differentiation of monocytes into dendritic cells is promoted by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon) and recombinant IL-4 (1000U/ml, R&D Systems) for 5 days.

Day 1: seed cells

Trypsinize cells and fix in 4 % PFA (or formalin)

Day 4: harvesting (virus-containing)

Day 3: change medium and start selection for stably transduced cells

Coculture of DCs and cells to be taken up (1(-2)x10^5 + 1(-2)x10^5 cells in 12-well) for 4 h to 24 h at 37°C

Staining of D2SC1/Flt3-DC and staining of the cells to be taken up by Mini67-1KT or Mini26-1KT (PKH67/PKH26 green/red fluorescent cell linker mini kit for general cell membrane labelling) from Sigma

Staining of GM-DC/Raw with anti-CD11c antibodies

Staining of the cells in PEalphaDC80 in PBS + 2 % FCS

Day 2: transfection

Day 1: 4 x 10^6 293 FT in 10 ml DMEM (+10 % FCS (Tet-frei), +P/S) in a 10cm plate