Abstract
In response to central nervous system (CNS) injury and infection, astrocytes, neurons, and CNS vasculature express several chemokines, including CCL21. Quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemical methods can quantify mRNA and protein expression. However, these methods do not quantify chemokine bioavailability and bioactivity, variables modified by many environmental factors including composition of the extracellular matrix (ECM). Here we illustrate how two-photon microscopy and carboxyfluorescein succinimidyl ester (CFSE or CFDA SE) labeling of T cells coupled with flow cytometry can be used as tools to assess chemokine-mediated regulation of T cell proliferation, activation, and migration.
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Acknowledgements
Assay development and data generation are supported by a University of California—Riverside Chancellor’s strategic initiative and National Institutes of Health grants NS071160A and NS072298A. The authors acknowledge Corinne Ploix for contributions toward standardization of flow cytometric protocols and Sean Wilson and Monica Rubalcava (Loma Linda Univerisity School of Medicine) for contributions toward optimization of multiphoton microscopy protocols.
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Carson, M.J., Wilson, E.H. (2013). Visualizing Chemokine-Dependent T Cell Activation and Migration in Response to Central Nervous System Infection. In: Cardona, A., Ubogu, E. (eds) Chemokines. Methods in Molecular Biology, vol 1013. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-426-5_11
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DOI: https://doi.org/10.1007/978-1-62703-426-5_11
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