Comparison of mature MDDC derived from fresh PBMC vs. cryopreserved PBMC of healthy donors. A. Surface phenotype: Mature MDDC derived from fresh or cryopreserved PBMC were stained with antibodies to CD209, CD86, CD83, and HLA-DR as described in Methods. For flow cytometric analysis, a gate was set on the cells with large scatter (size) that were expressing the myeloid DC specific marker CD209. The staining intensities (mean fluorescence intensity, MFI) of CD86, CD83, and HLA-DR were compared between mature MDDC derived from fresh or cryopreserved PBMC. B. Functional markers: Mature MDDC derived from fresh or cryopreserved PBMC were cultured for additional 18–20 h in presence of secretion inhibitor BFA. As described in Methods, cells were surface stained with antibodies to CD209, CD14, or CD86, and stained with antibodies to IL-12 and COX-2 for intracellular detection. For flow cytometric analysis, a gate was set on the large cells that also expressed CD209. Results are expressed as percentage of CD209+ cells that were positive for IL-12 (%CD209+IL-12+) or COX-2 (%CD209+COX-2+). Amounts of IL-8 (pg/ml) secreted by mature MDDC from each group were detected by using Cytometric Bead Array (CBA) technology (see Methods). Reported quantities (pg/ml) of the cytokines and chemokines reflect the production by 5 × 105 cells cultured in 3.75 ml medium. C. T cell stimulation: Scatter plot in the top panel shows proliferation of allogeneic CD4+ T cells using mature MDDC from fresh and cryopreserved PBMC of healthy donors. One to 2 × 105 MDDC were mixed with CFSE-labeled allogeneic fresh PBMC at a DC:PBMC ratio of 1:5 in a total volume of 1 ml/well of a 24-well plate. The lower two scatter plots demonstrate enhancement of MDDC mediated SEB-specific autologous CD4+ and CD8+ T cell proliferation. CFSE-labeled autologous PBMC from either fresh or cryopreserved healthy donors were added to the wells containing SEB alone or SEB-pulsed respective autologous mature MDDC at a DC:PBMC ratio of 1:5 as described in Methods. After four days of culture, cells were surface stained with CD3 PE, CD209 PerCP-Cy5.5 and CD4 APC and acquired on a flow cytometer. CD3+CD4+ lymphocytes were gated including the blasts and excluding CD209+ MDDC. The percentage of cells showing decreased CFSE staining intensity was reported as %proliferation. Bars in all the scatter plots represent medians. *, statistically significant differences (P < 0.05); **, statistically significant differences (P < 0.01).