Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates
- 272 Downloads
Detecting, imaging, and monitoring cell function on a single-cell basis is very important in the field of immunology research where many molecules are secreted from cells in response to external stimuli including immunization. Here, we introduce substrates with plasmonic nanoparticles and fluorescence microscopy as promising imaging methods for studies on molecular processes controlling cell behavior, particularly secretion of cytokines. We developed a unique composition of silver and silica layers of plasmonic nanostructures which resulted in fluorescence enhancement of more than 200-fold for the ensemble of molecules in the immunoassay. For the proof of concept demonstration, we used primary mouse macrophages and imaged tumor necrosis alpha secretion after stimulation of the cells with lipopolysaccharide. We demonstrate that metal-enhanced fluorescence assay provides imaging capability detection of cytokine secretion from a single cell without extensive biochemical procedures as required with standard methods. In addition, it is demonstrated that cell viability can be controlled during secretion.
KeywordsPlasmonic nanostructures Silver nanoparticles Metal-enhanced fluorescence Microphage cells Cell assays Tumor necrosis factor alpha
This research was supported by NIH grants R21CA147975, HG005090, HG002655, and AI0082299.
- 18.Thorpe, R., Wadhwa, M., Page, C., Mire-Sluis, A. (1999). Biological characterization and assay of cytokines and growth factors. Development in Biological Standarization, 97, 61–71.Google Scholar
- 19.Moody, M. D., Van Arsdell, S. W., Murphy, K. P., Orencole, S. F., Burns, C. (2001). Array-based ELISA for high-throughput anlysis of human cytokines. Biotechniques, 31(1), 186–196.Google Scholar
- 20.Becher, B., Blain, M., Giacomini, P. S., Antel, J. P. (1999). Inhibition of Th1 polarization by soluble TNF receptor is dependent on antigen-presenting cell-derived IL-12. Journal of Immunology, 162, 684–688.Google Scholar
- 25.Tanguay, S., Killion, J. (1994). Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine and Cytokine Research, 13, 259–263.Google Scholar
- 26.Boulet, S., Ndongala, M. I., Peretz, Y., Boisvert, M. P., Boulassel, M. R., Tremblay, C., Routy, J. P., Sekaly, R. P., Bernard, N. F. (2007). A dual color ELISPOT method for the simultaneous detection of IL-2 and IFN-γ HIV-specific immune responses. Journal of Immunological Methods, 320, 18–29.CrossRefGoogle Scholar
- 27.Karulin, A. Y., Hesse, M. D., Tary-Lehmann, M., Lehmann, P. V. (2000). Single-cytokine producing CD4 memory cells predominate in type 1 and type 2 immunity. Journal of Immunology, 164, 1862–1872.Google Scholar
- 28.Gazagne, A., Claret, E., Wijdenes, J., Yssel, H., Bousquet, F., Levy, E., Vielh, P., Scotte, F., Le Goupil, T., Fridman, W. H., Tartour, E. (2003). A Fluorospot assay to detect single T-lymphocytes simultaneously producing multiple cytokines. Journal of Immunological Methods, 283, 91–98.CrossRefGoogle Scholar
- 29.Chen, D. S., Soen, Y., Stuge, T. B., Lee, P. P., Webere, J. S., Brown, P. O., Davis, M. M. (2005). Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray. PLoS Medicine, 2(10), e265. doi: 10.1371/journal.pmed.0020265.CrossRefGoogle Scholar
- 30.Shirai, A., Holmes, K., Klinman, D. M. (1993). Detection and quantitation of cells secreting IL-6 under physiological conditions in BALB/c mice. Journal of Immunology, 150, 793–799.Google Scholar