title: Optimizing dendritic cell preparation for fusion with melanoma cells.
abstract: BACKGROUND
  Fusion of dendritic cells (DCs) with melanoma cells could reinforce the antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach.
  
  
  OBJECTIVE
  To define the dosage of the stimulating factors as well as the induction condition for the optimal DCs preparation and cell fusion.
  
  
  METHODS
  DCs were generated from murine bone marrow cells, and cultured with four different concentrations of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were confirmed to be mature by detecting the expression of MHC-II, CD11c, CD80, and CD83 by flow cytometry. DCs-melanoma fusion cells were generated using polyethylene glycols (PEG) with different molecular weights and the fusion efficiency was detected by fluorescence-activated cell sorter (FACS).
  
  
  RESULTS
  The largest quantity of DCs was found when cells were cultured with 1000 U/ ml of GM-CSF and 500 U/ml of IL-4 (1.69 ± 0.04 ×10⁶ ml⁻¹, p<0.001 when compared with the other three groups). The expression levels of MHC-II and CD83 on day 7 after incubation were significantly lower than those on day 3 (MHC-II: p<0.001; CD83: p<0.001). The efficiency of cell fusion under induction of PEG-3000 was significantly higher than that of PEG-4000 (15.4 ± 0.56% vs. 11.1 ± 0.45%, p<0.001).
  
  
  CONCLUSIONS
  The largest quantity for mature DCs was stimulated with 1000 U/ml of GM-CSF and 500 U/ml of IL-4 and the highest fusion efficiency was under induction of PEG-3000.
authors.affiliation: Department of Orthopaedics, First Affiliated Hospital, Dalian Medical University, Dalian 116000, Liaoning Province, China,email: yangliyyyl@163.com.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Shouyu
authors.initials: S
authors.lastname: Wang
authors.forename: Peng
authors.initials: P
authors.lastname: Shi
authors.forename: Wei
authors.initials: W
authors.lastname: Qu
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002459:Cell Fusion; D045744:Cell Line, Tumor; D003713:Dendritic Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D016130:Immunophenotyping; D015847:Interleukin-4 / Q000494:pharmacology; D008546:Melanoma, Experimental / Q000276:immunology / Q000473:pathology*; D051379:Mice; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000954:Antigens, Surface; D015847:Interleukin-4; D011092:Polyethylene Glycols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor