Cytokine capture with beads in cytotoxicity assays in microwells

Sammanfattning: Cytokines are small, secreted proteins that are important for cell signalling in theimmune system. Interferon gamma (IFN-γ) is one of the most potent cytokines thatnatural killer (NK) cells of the innate immune system secrete with both antiviral,antibacterial, and antitumoral activity. Analysis of NK cells, such as that of secretionof IFN-γ, is important for studying the immune response to cancer and for developingeffective immunotherapies. In this master thesis project, a method was developedfor determining the amount of IFN-γ secreted by NK cells when being confinedwith cancer cells in deep microwells. Antibody-coated microbeads was used tocapture secreted IFN-γ, which was fluorescently labeled and detected by imaging usingfluorescence microscopy. Microbead seeding into small microwells for single cellassays and into large microwells for embedding of beads into 3D tumor spheroidswas investigated. An analytical model based on experimental standard curves wasdeveloped for straightforward quantification of the amount of bound IFN-γ, with ademonstrated detection down to 2.10−18 moles per bead. The detection of IFN-γ wasevaluated for primary NK cells stimulated by PMA/ionomycin for different incubationtimes. The secretion rate of IFN-γ by IL-2 activated NK cells under PMA/ionomycinstimulation was estimated at 184 molecules per second. IFN-γ detection was alsoevaluated in cell cytotoxicity assays where NK cells were confined over time togetherwith cancer cells in microwells. Both assays showed a successful detection of IFN-γ secretion, demonstrating the potential of the developed method for immune cellanalysis.