Antigen expression following fusion. (A) Immature DCs and melanoma (Mel Im) cells were subjected to electrofusion (using different parameters), as well as PEG fusion, and then put into culture. As controls, DCs were cultured alone or with the maturation-inducing cytokines TNF-alpha, IL-6, IL-1beta and PGE2. After 3 days, cells were harvested and stained with anti-HLA-DR-FITC, anti-CD83-PE, anti-CD86-FITC, anti-CD80-FITC or isotype-matched control mAbs. Additional staining was performed with anti-CD45-PerCP mAb to label DCs. Samples were analyzed by two-color flow cytometry gating for CD45-positive cells. To exclude staining artifacts, melanoma cells were added to the DC control sample for the staining procedure. (B) Dendritic and (Mel Im) melanoma cells were fused by electrofusion or PEG fusion and cultured in the absence and presence of TNF-alpha, IL-6, IL-1beta and PGE2 (to induce maturation). After 2 days, IL-12 (p40) secretion in the supernatant was determined by ELISA. Values are the mean of duplicates of one representative experiment (standard deviation<10%).