| Popis: |
Introduction: Cancer vaccines are a therapeutic approach to initiate an antigen specific cytotoxic immune responses against tumors. Cancer vaccines are composed by an antigen (tumor peptide) and adjuvant. A peptide in combination with adjuvants effectively activate dendritic cells (DCs), the most efficient antigen presenting cells in our immune system. DCs prime and activate CD8+ cytotoxic T cells which generates an antigen specific response.Aim: Developing an assay to study the internalisation rout of an antigen-adjuvant conjugate in human dendritic cells by using live cell imaging. Method: Immobilisation of cells is necessary for the ability to perform live cell imaging for several hours. The immobilisation ability of three coatings, collagen type I, fibronectin and matrigel, at different concentrations were evaluated by using live cell imaging in a fluorescence microscope. The potential induction of activation of the cells were evaluated by using flow cytometry and ELISA. Results: Immature DCs internalise antigen-adjuvant conjugate more efficiently than mature and activated DCs. Therefore, it is important that the coating do not induce activation. Cells must also be immobilised for the possibility of long term detection. Collagen type I immobilised cells and induced activation in all investigated concentrations. Fibronectin and matrigel had concentration-dependent abilities to immobilise the cells. Matrigel did not activate the cells whilst fibronectin was concentration dependent. Conclusion: Matrigel immobilise the cells which enables long term single cell imaging without activation. |
