2 ml of cryopreserving solution selleck chemicals llc containing 5% dimethyl sulfoxide (Bioniche Pharma USA, Lake Forest, IL, USA). Finally fully equipped DCs were packed into a sterile glass vial (4��107 cells/vial), sealed with a snap-cap, and stored at an ultralow freezer for >12 h. Quality control of dendritic cell vaccine Safety test For safety, endotoxin, germ-free and mycoplasma-free tests were performed according to the KFDA-approved JW CreaGene standard and test guidelines. Endotoxin was evaluated using gel-clot techniques. The endotoxin of the product should be less than 10 EU/ml per 1.2-ml vial. Mycoplasma test was performed by both direct culture and PCR methods using e-Myco? Mycoplasma PCR detection kit (Intron Biotechnology, Seongnam, Korea), which contains primer sets specifically designed to detect major contaminants of Mycoplasma in cell cultures such as M.

arginini, M. faucium, M. fermentans, M. hyorhinis, M. orale, and A. laidlawii as well as other broad spectrum of mycoplasma. Cell size and granularity During the differentiation from monocytes to dendritic cells, cell size and granularity increase. Based on these principles, the cell size and granularity of each DC vaccine were assessed by flow cytometric analysis. PBMCs were used for gating control. Phenotypic analysis The phenotype of DC vaccine was determined by flow cytometry using a FACSCalibur? flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The following monoclonal antibodies were used: i) fluorescein isothiocyanate-conjugated mouse antihuman IgG2a isotype control; ii) phycoerythrin-conjugated mouse antihuman IgG1 isotype control; iii) anti-CD14, anti-CD19, anti-CD40, anti-CD80, anti-D86, anti-HLA-ABC, and anti-HLA-DR (BD Pharmingen, San Diego, CA, USA).

Viability The viability of DC vaccine was assessed by propidium iodide (PI) staining. PI (BD Pharmingen) was added to a sample and kept in the dark at room temperature for 20 min. Cell viability was examined by flow cytometry using a FACSCalibur? (Becton Dickinson). Viability was represented as 100-[(PI+ of sample)?(PI+ of control)] (%). Lymphocyte proliferation assay One vial from each DC vaccine lot was used to test of T cell stimulation capacity according to the standard lymphocyte proliferation assay. T cells were isolated from cryopreserved PBMC using nylon wool column (Polysciences, Warrington, PA, USA).

Purified T cells (1��105) were cultured with serially diluted DC vaccine (starting from 1��104 cells to 0.33��103 cells) at 37��C for 5 days. T cell proliferation was assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, yellow tetrazole: MTT) assay following manufacturer��s protocol AV-951 (CellTiter 96 Non-Radioactive proliferation assay kit; Promega, Madison, WI, USA). R2 represent the standard curve of MTT assay for the validation of a data set.